WO1995009180A1 - Polypeptide ii d'activation des monocytes endotheliaux, constituant un mediateur d'activation de la reponse d'hotes - Google Patents

Polypeptide ii d'activation des monocytes endotheliaux, constituant un mediateur d'activation de la reponse d'hotes Download PDF

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Publication number
WO1995009180A1
WO1995009180A1 PCT/US1994/011085 US9411085W WO9509180A1 WO 1995009180 A1 WO1995009180 A1 WO 1995009180A1 US 9411085 W US9411085 W US 9411085W WO 9509180 A1 WO9509180 A1 WO 9509180A1
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WIPO (PCT)
Prior art keywords
emap
cells
polypeptide
protein
tumor
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PCT/US1994/011085
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English (en)
Inventor
David M. Stern
Matthias Clauss
Janet Kao
Mark Kayton
Steven K. Libutti
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The Trustees Of Columbia University In The City Of New York
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Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Priority to US08/360,821 priority Critical patent/US6228837B1/en
Priority to EP94930525A priority patent/EP0721463A4/fr
Priority to AU79615/94A priority patent/AU7961594A/en
Priority to JP7510465A priority patent/JPH09505987A/ja
Publication of WO1995009180A1 publication Critical patent/WO1995009180A1/fr
Priority to US09/851,026 priority patent/US6734168B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Ra ⁇ fr -miTiH of the Invention Tumor vasculature is uniquely subject to the influence of products derived from the neoplastic cells. This may underlie the altered reactivity of vessels in certain tumors to catecholamines (1) , tumor necrosis factor (TNF) , flavone acetic acid (2) , as well as other agents.
  • TNF tumor necrosis factor
  • flavone acetic acid (2) flavone acetic acid
  • this tumor is sensitive to TNF, and infusion of the cytokine at low concentrations results in vascular compromise localized to the neoplastic lesions with early thrombosis/hemorrhage in the vessels and increased vascular permeability, and later regression of the tumor (3-7) .
  • cultured meth A tumor cells are relatively insensitive to TNF (3,8) . This suggests that tumor-derived mediators, potentially acting at the level of the endothelium, a central regulator of vascular tone, permeability and thrombogenicity, could be important in host-tumor interactions.
  • a prominent characteristic of immunogenic tumors is the presence of an inflammatory infiltrate surrounding the neoplastic lesion (103) .
  • One potentially important mechanism through which tumors modulate the host response is through the production of cytokines activating host effector cells, including mononuclear phagocytes (MPs) , polymorphonuclear leukocytes (PMNs) , and endothelial cells (ECs) (4-7) .
  • MPs mononuclear phagocytes
  • PMNs polymorphonuclear leukocytes
  • ECs endothelial cells
  • vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) (6) which modulates properties of ECs, including growth and induction of the procoagulant cofactor tissue factor, and MPs, including cell migration and tissue factor expression (8-13) .
  • VPF/VEGF vascular permeability factor/vascular endothelial growth factor
  • MPs including cell migration and tissue factor expression (8-13) .
  • endothelial-monocyte activating polypeptides I and II were isolated (5,7).
  • EMAP II a novel -20 kDa polypeptide which has recently been cloned and is not a member of previously described cytokine/chemokine families, has multiple effects on ECs, MPs, and PMNs in vitro, and induces an acute inflammatory response upon subcutaneous injection into mice (7) .
  • This invention provides a purified endothelial monocyte activating polypeptide II (EMAP II) .
  • This invention further provides a method of obtaining purified endothelial monocyte activating polypeptide II (EMAP II) .
  • This invention provides a method of obtaining antibodies to purified endothelial monocyte activating polypeptide II (EMAP II) .
  • This invention provides a method of detecting the presence of purified endothelial monocyte activating polypeptide II (EMAP II) in a sample.
  • EMAC II purified endothelial monocyte activating polypeptide II
  • This invention also provides an effector cell activating protein comprising a polypeptide having an amino acid sequence wherein at least four amino acid residues are the same as RIGRIVT and are in the same relative positions.
  • This invention further provides a method of detecting the presence in a sample of effector cell activating protein.
  • This invention provides a method of treating a tumor in a subject comprising administering an effective dose of endothelial monocyte activating polypeptide II (EMAP ID . Description of the Figures
  • Figure 1 Effect of EMAP II on migration and division of bovine aortic endothelial cells in an in vitro wound model.
  • Confluent monolayers of BAE were stimulated to migrate and divide by removal of a ring fence creating a 5mm diameter wound, at the time of wounding monolayers were exposed to EMAP II or control medium for 24 hours. Following incubation monolayers were washed, fixed in 3.5% paraformaldehyde in phosphate buffered saline containing 0.1% Nonidet P-40 and nuclei were stained with Hoechst 33258.
  • Control monolayers migrating into the wound margin display normal interphase nuclei (upper panel) compared with those exposed to EMAP, in which there are many condensed, pyknotic (apoptotic) nuclei (lower panel) . Wound margin is to the left.
  • FIG. 2 Infusion of EMAP II in murine inflammatory model. Mice were given intravenous injections of vehicle alone or vehicle containing EMAP via the tail vein and sacrificed by humane methods at 4 hours post infusion. Tissues were fixed in 10% formalin, processed routine methods and sections stained with hematoxylin and eosin. Lung from mice injected with vehicle alone are unremarkable (upper panel) while those from mice exposed to EMAP display evidence of inflammation, mild edema, and cellular infiltrate (lower panel) .
  • Figure 3 Light micrographs of footpads inoculated with either EMAP II-derived peptide-albumin conjugates or albumin alone.
  • Mouse footpads were injected with either albumin alone (A) , albumin exposed to glutaraldehyde (B) , albumin-RIGRIVTAKY (C) , or albumin- ASRLDLRIGRIVTAKY (D) . Following 6 hrs, footpads were harvested, processed as described in the text, and stained with hematoxylin/eosin. Magnification: x350.
  • Figure 4 Murine and Human EMAP II cDNA and EMAP II Sequence Derived Therefrom.
  • Figure 7 Percentage of Mouse Mammary Carcinoma Demonstrating Gross Hemorrhage Six Hours After EMAP II + TNF Treatment
  • Figure 8 (A) Tumor Regression After EMAP II + TNF Treatment: Treated vs. All Controls; (B) Tumor Regression After EMAP II + TNF Treatment: Treated vs. H.T. EMAP II + TNF;
  • Fig. 11 Northern analysis of Meth A cell RNA for EMAP II. The size of the message was determined by comparison to the migration of markers. 5 ug of poly(A)+ RNA was loaded.
  • Fig. 12 Hydrophilicity ploy of murine EMAP II.
  • the hydropathy profile was generated by the method of Kyte and Doolittle (74) .
  • the NH2- terminal sequence obtained from EMAP II purified from the supernatant of Meth A cells is indicated (N ⁇ 2 terminus) , and the Asp at the putative cleavage site for post- translational processing is shown. No evidence of a hydrophobic signal peptide is observed. Hydropathy is plotted versus deduced amino acid residue number.
  • Fig. 13 Comparison of EMAP II deduced amino acid sequence with the indicated residues from von Willebrand antigen II (vWAg II; 59, 60), IL-8 (54, 55), and IL-1B (48). All sequences are human, and numbering is based on the precursor form of each. Identical residues in two or more sequences are boxed.
  • Fig. 14A FPLC Mono C chromatography of E. coli extracts from cultures transformed to overexpress EMAP II. Following sonication of the E. coli pellet in Tris-buffered saline, supernatants (-35 mg of protein in each case) were applied to FPLC Mono Q (HR 5/5) , the column was eluted with an ascending salt gradient, and fractions were monitored for absorbance at 280 nm and induction of tissue factor activity (each sample was diluted 1:100 for the latter) in
  • Fig. 14B FPLC Mono Q chromatography of E. coli extracts from mock-transformed (i.e. vector alone) control cultures. See description of
  • Fig. 15A SDS-PAGE of EMAP II. The pool of fractions with maximum activity eluting from FPLC Mono
  • Fig. 15B SDS-PAGE of EMAP II.
  • Mono Q-derived, gel- eluted protein from slices of the gel which induced tissue factor in ECs was subjected again to reduced (IT) or nonreduced (UJ and far right panel) SDS- PAGE (12.5%; e ug/lane) .
  • Protein in the gel was either visualized by silver staining (JJ and JJJ) , or gel-eluted material was incubated with ECs, and induction of tissue factor activity, using the Factor Xa formation assay, was studied (far right panel) .
  • Gel slices are aligned with the corresponding portion of the stained gel in lane III.
  • Fig. 15C Immunoblotting of EMAP II.
  • EMAP II purified by FPLC Mono Q and SDS-PAGE/gel elution was subjected to nonreduced SDS-PAGE (12%; 10 ug/lane) , electrophoretic transfer to nitrocellulose, and immunoreactive protein in the gel was visualized using rabbit anti- mature EMAP II amino-terminal peptide IgG (2 ug/ml) followed by peroxidase-conjugated goat anti-rabbit IgG (50 ng/ml; Sigma) .
  • Molecular mass markers depict the migration of simultaneously run standard proteins: ovalbumin (46 kDa) , carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa) (Amersham Corp.).
  • Fig. 16A Effects of EMAP II on ECs: elevation of
  • Fig. 16B Effects of EMAP II on ECs: release of vWF.
  • Fig. 16C Effects of EMAP II on ECs: expression of P- selectin on the EC surface (C) .
  • EC monolayers (10 4 cells/well) in M199 were incubated at 37°C with EMAP II as above, heat-treated EMAP II, or thrombin (2 units/ml) .
  • a radioligand binding assay was then performed by adding 12S I-murine monoclonal anti-human P-selectin IgG (100 ng/ml) alone or in the presence of excess unlabeled anti-human P-selectin IgG, as described in the text.
  • Data shown were normalized by arbitrarily assigning the value of 1 (i.e 100%) to 125 I-antibody binding observed in the presence of thrombin (the maximal response) , and represent mean ⁇ S.E. of triplicate determinations.
  • Fig. 17A Effects of EMAP II on ECs: induction of EC tissue factor. Confluent ECs (10 4 cells/well) were incubated with the indicated concentration of EMAP II for 6 h at 37°C, then tissue factor activity was measured by determining Factor Vila- dependent Factor Xa formation as described in the text. As indicated: actinomycin D (5 ug/ml) was added simultaneously with EMAP II (star) : heat-treated EMAP II was adden place of active EMAP II (square) : or anti-tissue factor IgG (anti-TF: 1 ug/ml) was added during the tissue factor assay (triangle) . Data are reported as Factor Xa formed/min in the assay (ng/min) . Note that addition of the same amount of nonimmune IgG had no effect on Factor Xa formation in the tissue factor assay. The mean of duplicate determinations is shown, and the experiment was repeated four times.
  • Fig. 17B Effects of EMAP II on ECs: induction of EC tissue factor.
  • EC monolayers (as in Figure
  • Fig. 17C Effects of EMAP II on ECs: induction of EC tissue factor. ECs were incubated with medium alone (control) , LPS (100 ng/ml) , or EMAP II (100 pM) for 1 h and RNA was processed for amplification by PCR using primers for tissue factor (TF) or GAPDH. PCR was performed for 35 and 20 cycles, respectively. Migration of markers in base pairs ( X174) is shown on the far left lane.
  • Fig. 17D Effects of EMAP II on ECs: induction of E- selectin expression. Confluent ECs (10 4 cells/well) were incubated with either medium alone (control) , LPS (100 ng/ml) , or EMAP II (at the indicated concentration) for
  • Fig. 17E Effects of EMAP II on ECs: induction of E- selectin expression.
  • E confluent ECs (10 5 cells/well) were exposed to medium alone (control), LPS (100 ng) , or EMAP II (lOOpM) for 4 h and were then prepared for nonreduced SDS-PAGE (4-15%) .
  • 100 ug of protein was loaded in each lane.
  • proteins were transferred to nitrocellulose and visualized using murine monoclonal anti-human E-selectin IgG and peroxidase-conjugated goat-anti-mouse IgG. The migration of one simultaneously electrophoresed standard protein
  • Fig. 18A Effect of EMAP II on human PMNs: elevation of cytosolic calcium. EMAP II or heat- treated EMAP II was added to the fura-2- loaded cells (2 x 10 7 ) , and [Ca 2+ ] , was measured as described in the text. This tracing is representative of four experiments.
  • Fig. 18B Effect of EMAP II on human PMNs: peroxidase generation.
  • PMNs (3 x 10 6 cells/ml) were incubated with either phorbol ester (1, 10 uM; 2, 5 uM; 3, 2.5 uM) , EMAP II (4, 150 pM;
  • Fig. 18C Effect of EMAP II on human PMNs: chemotaxis.
  • PMNs (10 4 cells) were added to the upper compartment of chemotaxis chambers, and the indicated stimulus was added to the upper or lower compartment ⁇ upper /lower) .
  • fMLP (10" 6 ) or heat-treated assays were performed, and mean ⁇ S.E. is shown. * denotes p ⁇ 0.005 and ** denotes p ⁇ 0.001 compared with wells containing medium alone.
  • Fig. 19A Effect of EMAP II on MPs: expression of TNF. MPs (10 5 cells/well) were incubated with EMAP
  • Fig. 19B Effect of EMAP II on MPs: expression of TNF.
  • MPs (5 x 10 s ) were incubated with medium alone (control), LPS (100 ng) , EMAP II (150 pM) , or heat-treated EMAP II (H.T. EMAP II; 150pM) , and RNA was processed for amplification by PCR using primers for TNF or GAPDH. PCR was performed for 30 and 20 cycles for TNF and GAPDH, respectively. Migration of markers ( X174) base pairs is shown on the far left lane.
  • Fig. 19C Effect of EMAP II on MPs: expression of IL- 8.
  • MPs 105 cells/well
  • EMAP II 150 pM
  • LPS 100 ng
  • heat-treated EMAP II H.T. EMAP II: 150 pM
  • Supernatants were analyzed by ELISA for IL-8 antigen. The mean ⁇ S.E. of triplicate determinations is shown, and * denotes p ⁇ 0.004 and ** denotes p ⁇ 0.001 versus control.
  • Fig. 19D Effect of EMAP II on MPs: IL-8.
  • MPs (10 5 cells/well) were incubated with medium alone, LPS (100 ng) or EMAP II (150 pM) for 2 h, and RNA was processed for amplification by PCR using primers for IL-8 or GAPDH. PCR was performed with thermocyte settings described in the text. Migration of markers in base pairs is shown on the far left lane.
  • Fig. 19E Effect of EMAP II on MPs: chemotaxis.
  • MPs (10 3 cells/well) were added to the upper compartment of chemotaxis chambers and the indicated stimulus (concentration in pm) was added to the upper or lower compartment (upper/lower) .
  • fMLP (10 -6 ) or heat-treated EMAP II (100 pM) were added only to the lower compartment.
  • Cell migration assays were performed, and mean + S.E. is shown. * denotes p ⁇ 0.05 and ** denotes p ⁇ 0.002 compared with wells containing medium alone.
  • Fig. 19F Effect of EMAP II on MPs: expression of tissue factor.
  • MPs (5 x 10 4 cells/well) were incubated with EMAP II (100 pM) for the indicated times at 37°C, and then the tissue factor assay was performed and Factor Xa formation (mean ⁇ S.E. of triplicates) is shown.
  • * denotes MPs incubated with EMAP II (100 pM) for 2 h which were assayed for tissue factor activity in the presence of anti-tissue factor IgG (1 ug/ml) .
  • Fig. 19G Effect of EMAP II on MPs: expression of tissue factor.
  • MPs (5 x 10 5 ) were incubated with medium alone (control) , EMAP II (150 pM) , or LPS (100 ng) for 1 h, and RNA was processed for amplification by PCR using primers for tissue factor (TF) or GAPDH. PCR was performed for 40 and 25 cycles, respectively. Migration of markers in base pairs is shown on the far left lane.
  • Fig. 19H Effect of EMAP II on MPs: ionized cytosolic calcium.
  • EMAP II 200 pM was added to fura-2-loaded cells (2 x 10 7 ml) , and [Ca 2+ ] , was measured as described in the text. Each tracing is representative of four experiments.
  • Fig. 20A Systemic infusion of EMAP II into C3H/HeJ mice: cytokine induction. Mice were infused via the tail vein with EMAP II (10 ug) , and at the indicated times mice were sacrificed and anticoagulated blood samples were obtained. Plasma samples were assayed for IL-la ⁇ half -filled box) , IL-6 ⁇ open triangle) , or TNF ⁇ (open box) . The mean of duplicate determinations is shown, and this experiment is representative of four.
  • Fig. 20B Systemic infusion of EMAP II into C3H/HeJ mice: pulmonary leukostasis. Mice were infused with EMAP II (5 ug/animal) or saline, and at the indicated times lung tissue was harvested and myeloperoxidase activity was determined as described in the text. The mean + S.E. of triplicate determinations is shown, and the experiment was repeated three times.
  • Fig. 20C Systemic infusion of EMAP II into C3H/HeJ mice: athologic lung changes. Mice were infused with saline alone and then 4 h later were sacrificed; lung tissue was harvested, fixed, and stained with hematoxylin/eosin. x300 magnification.
  • Fig. 20D Systemic infusion of EMAP II into C3H/HeJ mice:pathologic lung changes. Mice were infused with EMAP II (10 ug) in saline, and then 4 h later were sacrificed; lung tissue was harvested, fixed, and stained with hematoxylin/eosin. x300 magnification.
  • Fig. 21A Intratumor injection of EMAP II into Meth A sarcomas: induction of hemorrhagic changes.
  • Meth A cells were administered by a single intratumor injection (0.1 ml) of either TNF
  • mice were sacrificed, and tumors dissected and graded for the presence or absence of hemorrhage.
  • Fig. 21B Intratumor injection of EMAP II into Meth A sarcomas: decrease in tumor volume (B) .
  • a tumors are beginning to spontaneously regress, as described previously (4) , although the degree of regression is less than that observed with tumors treated with
  • Fig. 22A Effects of combined cytokine treatments on murine mammary carcinomas: induction of hemorrhage.
  • Fig. 22B Effects of combined cytokine treatments on murine mammary carcinomas: induction of hemorrhage.
  • Mammary carcinomas (as in Fig. 22A above) received an intratumor injection of either EMAP II (10 ug/tumor) or heat- treated EMAP II (10 ug/tumor) followed ⁇ 15 h later by a tail vein injection of either TNF (5 ug/animal) or heat-treated TNF (5 ug/animal) .
  • Mice were sacrificed 6 h after the tail vein injection, and tumors were scored for the presence/absence of gross hemorrhage.
  • Fig. 22C Effects of combined cytokine treatments on murine mammary carcinomas: pathologic changes in the tumor bed.
  • Mammary carcinomas prepared as above
  • TNF 5 ug/animal
  • Fig. 22D Effects of combined cytokine treatments on murine mammary carcinomas: pathologic changes in the tumor bed.
  • Mammary carcinomas (prepared as above) received an injection of EMAP II (10 ug/tumor; D) , followed by tail vein injection if TNF (5 ug/animal) . Twelve h after TNF injection, tumors were excised, fixed, and stained as described. Magnification: x300.
  • Fig. 22E Effects of combined cytokine treatments on murine mammary carcinomas: reduction in clonogenic survival of tumor cells.
  • Tumors were excised 72 h later, processed to obtain diluted cell suspensions, counted, and incubated for 4 days in culture medium as described. After this incubation period, the number of dividing colonies was counted by a blinded observer and the surviving clonogenic fraction calculated.
  • Fig. 22F Effects of combined cytokine treatments on murine mammary carcinomas: and reduction in tumor volume.
  • Mammary carcinomas (grown as above) were injected locally with either EMAP II (10 ug/tumor) or vehicle alone, followed ⁇ 15 h later by tail vein infusion of TNF (5 ug/animal) or EMAP II (20 ug/animal) .
  • Tumor volume was evaluated on days 0 (the day of the tail vein injection) , 1, 3, and 7, and data are reported as percent initial tumor volume (calculated for each tumor and displayed as mean + S.E. for each treatment group) .
  • Statistical comparisons were made by ANOVA with post-hoc Student's t tests.
  • A Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  • TNF tumor necrosis factor
  • vWF von Willebrand Factor
  • PCR polymerase chain reaction
  • EC endothelial cell
  • EMAP endothelial-monocyte activating polypeptide
  • VPF/VEGF vascular permeability factor/vascular endothelial growth factor
  • GAPDH glyceraldehyde phosphate dehydrogenase
  • fMLP formyl-methionyl- leucinyl-phenylalanine
  • PMN polymorphonuclear leukocyte
  • MP or mononuclear mononuclear phagocyte
  • IL interleukin
  • IL-1 interleukin 1
  • Meth A methylcholanthrene A-induced murine fibrosarcoma
  • TMB 3,3' ,5,5' -tetramethylbenzidine
  • DSS disuccinimidyl suberate
  • cytosolic free calcium concentration cytosolic free calcium concentration.
  • This invention provides a purified endothelial monocyte activating polypeptide II (EMAP II) .
  • This invention further provides an endothelial monocyte activating polypeptide II (EMAP II) having an apparent molecular weight of about 20,000 Daltons. More particularly, the EMAP II has an apparent molecular weight between about 18,000 Daltons and about 22,000 Daltons.
  • EMAP II endothelial monocyte activating polypeptide II
  • endothelial monocyte activating polypeptide is murine endothelial monocyte activating polypeptide (EMAP II) .
  • endothelial monocyte activating polypeptide II comprises the sequence Gly-Lys-Pro-Ile-Asp-Ala-Ser-Arg-Leu-Asp-Leu- Arg-Ile-Gly-Xaa-Ile-Val-Thr-Ala-Lys.
  • Gly-Lys-Pro-lie-Asp-Ala-Ser-Arg-Leu-Asp- Leu-Arg-Ile-Gly-Xaa-Ile-Val-Thr-Ala-Lys is thesequence of the N-terminal twenty amino acid residues.
  • This invention provides an antibody capable of binding to endothelial monocyte activating polypeptide II.
  • This antibody may be a polyclonal antibody. Alternatively, it may be a monoclonal antibody.
  • This invention further provides a method of obtaining purified endothelial monocyte activating polypeptide II comprising, a) obtaining conditioned medium containing Meth A cells; b) purifying the medium from Meth A cells; c) applying the purified medium to a cation exchange resin; d) step-eluting from the cation exchange resin and pooling fractions with OD 280 > 0.05; e) applying the pooled fractions to an FPLC column; and f) eluting with an ascending salt gradient, thereby obtaining purified endothelial monocyte activating polypeptide II.
  • This invention also provides a method of obtaining an antibody to purified endothelial monocyte activating polypeptide II comprising a) immunizing a rabbit with Gly-Lys-Pro-Ile-Asp-Ala-Ser-Arg-Leu-Asp-Leu-Arg-Ile- Gly-Cys-lie-Val-Thr-Ala-Lys coupled to keyhole limpet hemocyanin; and b) obtaining purified IgG from the rabbit.
  • the antibody is a polyclonal antibody.
  • This invention provides a method of detecting the presence in a sample of EMAP II comprising a) adding cells to a to a first chamber; b) adding the sample to a second chamber which is separated from the first chamber by a membrane; c) visualizing migrating cells; d) counting the migrating cells; and e) determining the presence of EMAP II.
  • the cells are mononuclear phagocytes. In another embodiment, the cells are polymorphonuclear leukocytes.
  • This invention also provides a method of detecting the presence in a sample of EMAP II comprising a) injecting the sample into an animal footpad; and b) detecting an inflammatory response, thereby indicating the presence of EMAP II.
  • the animal footpad is a mouse footpad.
  • This invention also provides a method of detecting the presence in a sample of EMAP II comprising an immunoprecipitation step.
  • This invention also provides a method of detecting the presence in a sample of EMAP II comprising a) contacting cells with the sample; and b) assaying for tissue factor activity, thereby indicating the presence of endothelial monocyte activating polypeptide II.
  • the cells are endothelial cells.
  • the cells are monocytes.
  • This invention also provides a method of inducing chemotaxis comprising a) adding cells to a to a first chamber; and b) adding a chemotaxis-inducing effective amount of EMAP II to a second chamber which is separated from the first chamber by a membrane, thereby inducing chemotaxis of the cells.
  • the cells are mononuclear phagocytes.
  • the cells are polymorphonuclear leukocytes.
  • This invention provides a method of inducing inflammation in a subject comprising injecting an inflammation-inducing effective amount of endothelial monocyte activating polypeptide II into the footpad of the subject.
  • the subject is a mouse.
  • This invention also provides a method of inducing tissue factor comprising contacting cells with a tissue factor-inducing effective amount of endothelial monocyte activating polypeptide II.
  • the cells are endothelial cells.
  • the cells are monocytes.
  • This invention further provides an effector cell activating protein comprising a polypeptide having an amino acid sequence wherein at least four amino acid residues are the same as Arg-Ile-Gly-Arg-Ile-Val-Thr (RIGRIVT) and are in the same relative positions.
  • AILRQVT has at least four amino acid residues that are the same as RIGRIVT and in the same relative positions because AILRQVT matches RIGRIVT in positions 2, 4, 6 and 7.
  • the protein may have any number of amino acid residues as long as any seven-residue segment of the protein has at least four residues that are the same as RIGRIVT and in the same positions relative to each other.
  • LAILRQVT has four residues that are the same as RIGRIVT and are in the same relative positions because LAILRQVT matches RIGRIVT at positions 3, 5, 7 and 8 of LAILRQVT.
  • RGRIVTI has all residues the same as RIGRIVT but only one residue is in the same relative position because RGRIVTI matches RIGRIVT only in position 1.
  • at least five amino acid residues are the same as RIGRIVT and are in the same relative positions.
  • at least six amino acid residues are the same as RIGRIVT and are in the same relative positions.
  • a more specific embodiment comprises RIGRIVT.
  • the effector cell activating protein has at least seven amino acids. In a further embodiment the effector cell activating protein has between about 7 and about 16 amino acids.
  • the effector cell activating protein is labeled.
  • the label is a radioactive label.
  • the radioactive label is 15 I.
  • the effector cell activating protein comprises a polypeptide having an amino acid sequence selected from the group consisting of:
  • ASRLDLRIGRIVTAKY ASRLDLRIGRIVTAK
  • ASRLDLRIGRIVTAK ASRLDLRIGRIVTAK
  • LRIGRIVTAKY LRIGRIVTAKY
  • the effector cell activating protein is conjugated to an immobilizer.
  • the immobilizer preferably comprises a polypeptide having a molecular weight of at least about 5,000 daltons.
  • the immobilizer is albumin.
  • This invention provides an antibody capable of binding to the effector cell activating protein.
  • the antibody is a polyclonal antibody.
  • the antibody is a monoclonal antibody.
  • This invention further provides a method of obtaining an antibody to effector cell activating protein comprising a) immunizing a rabbit with the effector cell activating protein coupled to keyhole limpet hemocyanin; and b) obtaining purified IgG from the rabbit.
  • the antibody is a polyclonal antibody. In another embodiment, the antibody is a monoclonal antibody.
  • This invention provides a method of detecting the effector cell activating protein.
  • This invention provides a method of detecting the presence in a sample of effector cell activating protein comprising a) adding cells to a first chamber; b) adding the sample to a second chamber which is separated from the first chamber by a membrane; c) visualizing migrating cells; d) counting the migrating cells; and e) determining the presence of the effector cell activating protein.
  • the cells are ononuclear phagocytes.
  • the cells are polymorphonuclear leukocytes.
  • This invention also provides a method of detecting the presence in a sample of effector cell activating protein comprising the steps of a) injecting the sample into an animal footpad; and b) detecting an inflammatory response, indicating the presence in the sample of effector cell activating protein.
  • the animal footpad is a mouse footpad.
  • a specific embodiment of the method of detecting the effector cell activating protein comprises a step of detecting binding to mononuclear phagocytes.
  • This invention provides a method of detecting the effector cell activating protein comprising a step of detecting increased [Ca 2+ ]i in effector cells.
  • the effector cells are selected from the group consisting of mononuclear phagocytes and polymorphonuclear leukocytes.
  • This invention also provides DNA encoding the effector cell activating protein.
  • This DNA may comprise the coding strand or the strand complementary to the coding strand. It may be single-stranded or double-stranded, circular or linear. It may further comprise promoters and other expression control sequences known to one with skill in the art to which this invention pertains. Because of the degeneracy of the genetic code, which is well known to one with skill in the art to which this invention pertains, various DNA sequences code for a single amino acid sequence.
  • this invention provides DNA encoding the effector cell activating protein which comprises an amine acid sequence selected from the group consisting of:
  • RIGRIVTAKY ASRLDLRIGCIVTAK; ASRLDLRIGRIVTAKY; ASRLDLRIGR ⁇ VTAK,• LRIGRIVTAKY;
  • This invention provides a method of using the effector cell activating protein to induce cell chemotaxis.
  • the cells are mononuclear phagocytes.
  • the cells are polymorphonuclear leukocytes.
  • This invention provides a method of inducing chemotaxis comprising a) adding cells to a first chamber; and b) adding a chemotaxis-inducing effective amount of the effector cell activating protein of claim 27 to a second chamber which is separated from the first chamber by a membrane, thereby inducing chemotaxis of the cells.
  • the cells are mononuclear phagocytes.
  • the cells are polymorphonuclear leukocytes.
  • This invention further provides a method of inducing inflammation in a subject comprising administering an inflammation-inducing effective amount of the effector cell activating protein.
  • This invention also provides a method of increasing [Ca 2"1" ]; in effector cells using the effector cell activating protein.
  • the effector cells are selected from the group consisting of mononuclear phagocytes and polymorphonuclear leukocytes.
  • This invention further provides a method of treating a tumor in a subject comprising administering an effective dose of endothelial monocyte activating polypeptide II (EMAP II) .
  • EMP II endothelial monocyte activating polypeptide II
  • this invention provides a method for treating the tumor by inducing hemorrhage in the tumor.
  • this invention provides a method for treating the tumor by reducing the volume of the tumor.
  • the volume of the tumor is reduced by at least twenty-five percent (25%) .
  • this invention provides a method of treating a methylcholanthrene A - induced fibrosarcoma tumor in a subject comprising administering an effective dose of endothelial monocyte activating polypeptide II (EMAP II) .
  • EMP II endothelial monocyte activating polypeptide II
  • the subject is a mammal. In a more specific embodiment the subject is a mouse. In another specific embodiment, the subject is a human.
  • this invention provides a method of treating a tumor in a subject comprising administering an effective dose of endothelial monocyte activating polypeptide II (EMAP II) wherein the effective dose is between about two micrograms and about fifty micrograms. In a more specific embodiment the effective dose is about twenty micrograms. In another specific embodiment the effective dose is between about six micrograms and about one hundred fifty micrograms. In a more specific embodiment the effective dose is about sixty micrograms.
  • An embodiment of the method for treating a tumor in a subject further provides that the endothelial monocyte activating polypeptide II (EMAP II) is in a pharmaceutically acceptable carrier.
  • administering comprises injecting intratumorally. In another embodiment, the administering further comprises administering systemically.
  • the tumor comprises carcinoma cells.
  • the carcinoma cells are mouse mammary carcinoma cells.
  • Another embodiment further comprises administering an effective dose of tumor necrosis factor.
  • the effective dose of tumor necrosis factor is administered systemically.
  • the effective dose is between about 500 nanograms and about fifteen micrograms.
  • the effective dose is about five micrograms.
  • this invention provides a method for treating a tumor in a subject wherein the tumor comprises carcinoma cells.
  • the carcinoma cells are mouse mammary carcinoma cells.
  • This invention further provides the method for treating a tumor in a subject wherein the EMAP II is recombinant EMAP II.
  • This invention further provides the method for treating a tumor in a subject wherein the endothelial monocyte activating polypeptide II (EMAP II) comprises: S PIDASRLDLRIGCIVTAKKHPDADSLYVEEVDVGEAAPRTWSGLV
  • This invention further provides a pharmaceutical composition comprising an effective amount of endothelial monocyte activating polypeptide (EMAP II) in a pharmaceutically acceptable carrier.
  • EMAP II endothelial monocyte activating polypeptide
  • Meth A cells provided by Drs. Hoffman and Old (Memorial Sloan-Kettering Cancer Center) (11) , were grown in RPMI 1640 containing 10% calf serum (Hyclone Labs, Logan, UT) using a continuously perfused three liter bioreactor (Bellco Biotechnology, Vineland, NJ) . The bioreactor was washed with ten liters of serum-free medium (to remove serum components from complete medium) , and then serum- free conditioned medium was collected at a rate of 416 ml/h, and concentrated 20-fold by ultrafiltration (12). Human umbilical vein endothelial cells (ECs) were prepared by the method of Jaffe (13) , as modified by Thornton et al (14) . Experiments were performed within 48 hours of the cells achieving confluence.
  • Jaffe Jaffe
  • Chizzonete Hoffmann-LaRoche
  • antibody to urine tumor necrosis factor was purchased from Genzyme (Cambridge, MA) .
  • Murine EMAP I and VPF/VEGF were prepared as described previously (9,22) .
  • Murine IL-l ⁇ was generously provided by Dr. P. Lomedico (Hoffmann- LaRoche) and murine TNF ⁇ was obtained from Genzyme (Cambridge, MA) .
  • a peptide based on the amino acid sequence was employed as immunogen.
  • the peptide comprised the N-terminal sequence with Cysteine substituted for the undetermined amino acid at position 15 (see Table 1) (Multiple Peptide Systems, San Diego, CA) and an additional cysteine at the carboxy terminus to facilitate coupling to keyhole limpet hemocyanin using M-maleimidobenzoyl-N-hydroxysuccinimide (21) .
  • Rabbits were immunized by standard methods (initial immunization: 1 mg/animal; monthly boosts: 500/xg/animal; intradermal) .
  • Rabbit IgG purified by affinity chromatography on protein A-Sepharose (Pharmacia) (22) , was screened by ELISA (Enzyme-Linked Immunosorbent Assay) using purified -22 kDa polypeptide.
  • the ELISA was performed as follows: partially purified -22 kDa polypeptide or purified -22 kDa polypeptide in coating buffer (Na 2 C0 3 , 15 mM; NaHC0 3 , 35 mM, CaCl 2 , 0.1 mM; final pH 9.2) were incubated overnight at 4°C in Nunc-Immuno Plate Maxisorp (Nunc-Kamstrup, Denmark) .
  • washing buffer Tris/HCl, 20 mM; NaCl, 120 mM; Tween 20, 0.05%; final pH 7.4
  • the primary antibody (3 ⁇ g/ml) was added for 1 hour at 3 °C, wells were washed 4 times with washing buffer, and then incubated with peroxidase conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) at a 1:1000 dilution for an additional 1 hour at 37°C.
  • Western blotting and immunoprecipitation of -22 kDa meth A factor Western blotting was performed using purified -22 kDa meth A-derived polypeptide, as well as other samples, by subjecting them to SDS-PAGE (12%) and electroblotting onto nitrocellulose paper (23) . Reactive sites on the nitrocellulose were blocked overnight at room temperature with 3% nonfat dry milk in tris-buffered saline containing Tween 20(0.05%) (24) .
  • nitrocellulose membranes were incubated for 2 hrs with polyclonal rabbit antibodies raised to the —22 kDa meth A factor (3 ⁇ g of immune IgG) . Sites of primary antibody binding were detected with a secondary antibody conjugated to horseradish peroxidase using a kit from Amersham
  • A-derived polypeptide was estimated from the migration of standard proteins run simultaneously: phosphorylase b, 97.4 kDa, bovine serum albumin, 69 kDa, ovalbumin 46 kDa, carbonic anhydrase, 30 kDa, trypsin inhibitor,
  • Immunoprecipitation was performed by labelling cells metabolically with 35 S-methionine as previously described (27) .
  • meth A cultures were incubated for 72 hrs in methionine-poor serum-free medium supplemented with 35 S-methionine (lO ⁇ Ci/ml) , supernatants were harvested, diluted 1:1 with 50 mM MES (pH 5.5), applied to FPLC Mono S (HR 5/5; Pharmacia), and the column was then eluted with an ascending salt gradient (0 to 1 M NaCl) .
  • IgGSorb formalin-fixed, protein A-bearing Staphylococcus aureus
  • the immune precipitate was washed four times with tris-buffered saline (tris/HCl, 20 mM; pH 7.4; NaCl, 120 mM) containing NP-40 (0.25%), non-reducing Laemmli buffer was added, and the sample was boiled prior to SDS-PAGE.
  • tris-buffered saline tris/HCl, 20 mM; pH 7.4; NaCl, 120 mM
  • NP-40 0.25%
  • Tissue factor activity in human ECs was assayed by incubating confluent cultures (9.6cm 2 growth area; -1.2xl0 5 cells/cm 2 ) with purified -22 kDa polypeptide in Medium 199 containing HEPES (10 mM; pH 7.4) , polymyxin B (50 units/ml) and fetal calf serum (5%) in the presence/absence of other agents, such as either cycloheximide (10 ⁇ g/ml) , actinomycin D (5 ⁇ g/ l) , antibodies to the -22 kDa factor, or VPF/VEGF.
  • HEPES 10 mM
  • polymyxin B 50 units/ml
  • fetal calf serum fetal calf serum
  • -22 kDa meth A factor was treated with trypsin (5 ⁇ g/ml for 2 hr at 37°C; trypsin was inactivated by addition of aprotonin, 25 ⁇ g/ml, Sigma) or heated (100°C for 10 min; this destroys -22 kDa meth A factor activity, but has no effect on endotoxin- mediated induction of tissue factor activity) prior to addition to endothelial cell cultures. Monolayers were then incubated for the indicated times at 37°C, cells were scraped into suspension with a rubber policeman, and tissue factor activity was determined using a coagulant assay, as described previously (9,22) .
  • tissue factor (2.5 ⁇ g/ml; generously provided by Dr. W. Kisiel, Univ. of New Mexico, Albuquerque, NM) was added to certain cell preparations just prior to performing the coagulant assay. Tissue factor equivalents were determined using a standard curve from experiments with purified human tissue factor (26) . Tissue factor reconstituted into phosphati- dylserine/phosphatidylcholine vesicles (20:80) was generously provided by Dr. Ronald Bach, Univ. of Minnesota, Minneapolis, MN) .
  • Procoagulant activity of mouse macrophages was determined as follows: suspensions of macrophages (10 4 cells/assay) , isolated from the peritoneum 3-4 days after stimulation with thioglycollate broth (2 ml; Sigma) , were incubated with -22 kDa meth A factor alone or in the presence of other agents for the indicated times at 37°C in RPMI 1640 containing HEPES (10 mM, pH 7.4), penicillin, streptomycin (50 U/ml; 50 ⁇ g/ml) , ⁇ - mercaptoethanol (5xl0" s M) , and polymyxin B (50 units/ml) .
  • Tissue factor was determined as described above using the coagulant assay with murine plasma. Data are expressed as clotting time in seconds per sample assayed, since purified murine tissue factor is not available to use as a standard.
  • tissue factor mRNA transcripts in human ECs and mononuclears exposed to -22 kDa meth A factor was studied using the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • total RNA was extracted from stimulated or quiescent cells using the guanidiniu thiocyanate procedure (27) .
  • First strand cDNA was synthesized with oligo dT primer (BRL, Bethesda, MD) and served as template for PCR analysis.
  • Tissue factor primers were generously provided by Dr. W. Konigsberg
  • Nonadherent cells were removed by washing the plate twice with balanced salt solution, and adherent cells were harvested by incubation with calcium-magnesium free buffer containing EDTA (2 mM) for 15 min at 37°C, followed by extensive washing. PMNs were prepared by centrifugation over Hisopaque 1119 as per the manufacturer's protocol (Sigma) . Chemotaxis assays were performed in microchemotaxis chamber (NeuroProbe, Bethesda, MD) containing Nucleopore polycarbonate membranes (5 ⁇ m; Nucleopore, Pleasonton, CA) .
  • Mononuclears or PMNs were suspended in RPMI 1640 containing fetal bovine serum (1%) and 10 4 cells were added per well to the upper chamber.
  • the chemotactic stimulus was added to the indicated chamber, and assays were performed in quadruplicate over a 3 hr or 45 min incubation period at 37°C, with mononuclear cells or PMNs, respectively, after which non-migrating cells were removed, membranes were fixed in methanol, migrating cells were visualized with Wright's stain. Cells in nine high-power fields were counted, and the mean and standard error of the mean (SEM) were determined.
  • mice Female Balb/c mice (6-12 wks) were injected with -0.03 ml of either (i) Tris-buffered saline, -22 kDa meth A polypeptide (homogeneous, gel-eluted material), (ii) gel-eluted material from a region of the same SDS gel which had no -22 kDa meth A factor, (iii) V6,13 22 kDa meth A factor which had been pre-treated with trypsin (enzyme:substrate ratio, 1:50, w:w) for 1 hr at 37°C followed by addition of aproptonin (0.5 «*g), or (iv) -22 kDa meth A factor was heat-treated at 100°C for 10 min to destroy tryptic activity.
  • trypsin enzyme:substrate ratio, 1:50, w:w
  • aproptonin 0.5 «*g
  • footpad thickness was measured with calipers (each footpad was measured five times at each time point) , and, subsequently, animals were sacrificed. Footpads were fixed in buffered formalin (10%) , decalcified, and embedded in paraffin. Sections were stained with hematoxylin and eosin.
  • the current report defines a third, novel meth A-derived polypeptide, distinct from those previously studied, which modulates endothelial and white cell functions.
  • the column was resolved with an ascending salt gradient, leading to the definition of three major peaks of activity, assessed by the induction of tissue factor activity in cultured ECs.
  • the pool of fractions in activity peak I provided starting material for purification of EMAP I, a polypeptide with Mr -40,000, which was previously identified in tumor-conditioned medium (9,10).
  • the material in activity peak III was used for preparation of murine Vascular Permeability Factor/Vascular Endothelial Growth Factor (VEGF/VPF) , and its activity could be neutralized by polyclonal antibody to guinea pig VPF, as described previously (22) .
  • VEGF/VPF Vascular Permeability Factor/Vascular Endothelial Growth Factor
  • Activity peak II from the Mono S column was further analyzed by nonreducing SDS-PAGE, and elution of protein from nitrocellulose membranes after Western blotting. Although the pattern of protein bands visualized by Coomassie blue staining of the gels was complex, as expected form the chromatogram of the Mono S column, there were only two areas on the gel, corresponding to Mr -40,000 and -22,000, which on elution had the capacity to induce tissue factor activity in ECs. Since the higher molecular weight material was likely to correspond to EMAP I or VPF/VEGF, our attention was focussed on the factor(s) responsible for the activity at Mr -22,000.
  • the -22 kDa polypeptide was characterized structurally, by N- terminal sequencing, and immunologically, using an antiserum prepared to a peptide comprising the N- terminal sequence, in order to assess its relationship to other mediators present in the tumor-conditioned medium.
  • the portion of the vWF antigen II sequence shown corresponds to Asp (480) to Ser (490) , and was deduced from the cDNA (32-33) .
  • Antibodies to the -22 kDa polypeptide were prepared by immunizing rabbits with a synthetic peptide comprising the amino terminal sequence coupled to keyhole limpet hemocyanin. IgG from this antiserum neutralized the ability of the -22 kDa meth A factor to induce tissue factor activity in ECs in a dose-dependent manner and adsorbed the activity when the antibody was bound to a solid support. In contrast, non-immune IgG was without effect.
  • Immunoblotting with IgG prepared to the synthetic peptide following non-reduced SDS-PAGE, visualized a major band with Mr 22,000 in samples of purified -22 kDa meth A factor and fractions from activity peak II from FPLC Mono S. Shorter exposure times of blots to the film showed that this major band was composed of two closely migrating bands. Addition of excess purified -22 kDa meth A factor during incubation of blots with the anti-peptide antibody greatly diminished intensity of the band, indicating that the antibody was recognizing determinants on EMAP II.
  • the IgG fraction of antiserum to the amino terminal peptide derived from the —22 kDa meth A factor was employed to construct an ELISA.
  • This ELISA was used to monitor the purification procedure of the —22 kDa polypeptide (Table 2) : about 195-fold purification was required to obtain homogeneous -22 kDa meth A factor with the series of steps used.
  • *EMAP II antigen was measured using an ELISA, as described in the text.
  • the starting volume of culture supernatant for this preparation was about 40 liters.
  • the level of transcripts for tissue factor mRNA increased on exposure to -22 kDa meth A factor, as indicated by the greater intensity of the PCR reaction product.
  • the level of transcripts for glycerceraldehyde phosphate dehydrogenase (GAPDH) mRNA in ECs was unchanged under these conditions.
  • EMAP II- mediated induction of EC tissue factor was not likely to be due to contaminating endotoxin, as demonstrated by the inhibitory effect of antibody raised to the amino terminal EMAP II peptide and pre-treatment of the polypeptide with trypsin.
  • all assays of endothelial procoagulant activity were performed in the presence of polymyxin B.
  • Mononuclear cells associated with tumors are often enmeshed in fibrin, suggesting that they might express procoagulant activity (38) . Therefore, experiments were performed to examine if -22 kDa meth A factor could induce monocyte procoagulant activity.
  • Incubation of murine peritoneal macrophages with EMAP II resulted in induction of procoagulant activity, as shown by the ability of the treated cells to shorten the clotting time of recalcified murine plasma.
  • Induction of procoagulant activity occurred in a time- dependent manner, peaking at about 6-12 hours, and could be blocked almost completely by a monospecific antibody against human tissue factor, indicating that most of the clot promoting activity was due to tissue factor.
  • Tissue factor expression by mononuclears in response to -22 kDa meth A factor was also dependent on the polypeptide's concentration, could be blocked by treating EMAP II with trypsin, and required the integrity of biosynthetic mechanisms, as it was prevented by addition of actinomycin D to cultures . Similar to the results on ECs described above, enhanced expression of mononuclear cell tissue factor activity was accompanied by an increase in the level of tissue factor mRNA transcripts, as evidenced by PCR.
  • Immunogenic tumors such as the meth A fibrosarcoma
  • Experiments were performed to examine if the -22 kDa meth A polypeptide could induce migration of human PMNs and mononuclear cells harvested from peripheral blood (Tables 3-4) .
  • Experiments in microchemotaxis chambers demonstrated that EMAP II enhanced cell migration in a dose-dependent manner for PMNs (Table 3) and for mononuclear cells (Table 4) .
  • Cell migration in response to EMAP II was prevented by exposing the polypeptide to trypsin or by adsorption of EMAP II with polyclonal antibody to the N-terminal peptide.
  • ⁇ Cell migration assays were performed by adding PMNs to the upper wells of microchemotaxis chambers, and placing the indicated concentration of — kDa meth A factor in the upper and/or lower wells. The incubation period was 45 min at 37°C. Migrating cells from nine representative high-powered fields are shown (the mean and standard deviation, S.D.) .
  • ⁇ Cell migration assays were performed by adding mononuclear cells to the upper wells of microchemotaxis chambers, and placing the indicated concentration of -22 kDa meth A factor in the upper and/or lower wells. The incubation period was 3 hr at 37°C. Migrating cells from nine representative high-powered fields are shown (mean and standard deviation, S.D.).
  • Phlogogenic properties of —22 kDa meth A-derived polypeptide (EMAP II) in the mouse footpad model Phlogogenic properties of —22 kDa meth A-derived polypeptide (EMAP II) in the mouse footpad model.
  • EMAP II meth A-derived polypeptide
  • swelling was observed as evidenced by the increase in footpad thickness compared with buffer controls.
  • the footpad showed an acute inflammatory response characterized by a PMN infiltrate and edema in the subcutaneous tissues, compared with the untreated control.
  • the inflammatory response had begun to recede by 8 hr after injection of EMAP II.
  • trypsin-treatment of polypeptide abrogated its phlogogenic properties (Figure 2) .
  • Immunogenic tumors such as the murine meth A fibrosarcoma, characteristically have a peripheral zone which contains a chronic inflammatory infiltrate (38- 41) .
  • the presence of these inflammatory cells often embedded in a meshwork of fibrin which can extend throughout the tumor stroma, contributes to the concept that tumors might be considered "wounds that do not heal (38) . " This has led us to identify tumor-derived mediators which could prime the host response, altering endothelial properties and attracting inflammatory cells to the tumor.
  • EMAP I a trypsin-sensitive, -40 kDa polypeptide distinct from other cytokines and growth factors (9-10)
  • a polypeptide which turned out to be the murine homolog of VPF/VEGF (22) a factor which had previously been shown to increase vascular permeability and to be mitogenic for ECs (42-46) .
  • EMAP II meth A tumor cells
  • EMAP II activates ECs and mononuclear cells, potentiating their participation in procoagulant reactions through induction of tissue factor, promoting migration of monocytes and PMNs, and leading to a phlogogenic response when injected into murine footpads.
  • EMAP II is an apparently unique polypeptide which runs as a broad band, Mr -22,000. In view of the apparently similar spectrum of biological properties of EMAP II and the other two mediators, VPF/VEGF and EMAP I, it was important to determine if there was a relationship between these molecules.
  • EMAP II amino terminal sequence and chromatographic properties of EMAP II were distinct, it could represent an alternatively spliced or degraded form derived from the other polypeptides, which appear to be about twice as large, Mr 38-44 kDa versus -22 kDa.
  • studies with polyclonal antibody directed against the amino terminal portion of EMAP II did not show any immunoreactivity with either EMAP I or VPF/VEGF.
  • polyclonal antibodies which adsorb murine VPF/VEGF (22) did not react with EMAP II (data not shown) .
  • metabolic labelling and immunoprecipitation of meth A tumor cells demonstrated EMAP II to be synthesized as a -22 kDa polypeptide, no larger precursor form was evident.
  • EMAP II has distinct biologic activities compared with EMAP I and VPF/VEGF: EMAP II stimulates PMN migration, in contrast to the other two mediators, but EMAP II does not directly increase EC monolayer permeability in culture, whereas EMAP I and VPF/VEGF do.
  • molecular cloning studies have shown EMAP II to be distinct from EMAP I, VPF/VEGF, and vWF antigen II. With respect to vWF antigen II, the region of sequence homology with EMAP II is limited to the portion of the amino terminus shown in Table 1.
  • EMAP II ENDOTHELIA -MONOCYTE ACTIVATING POLYPEPTIDE II MATERIALS AND METHODS Purification of EMAP II. preparation and radiolabelling of synthetic peptides.
  • Murine EMAP II (EMAP II) , purified as described (7) , was homogeneous on SDS-PAGE, migrating as single band, Mr -18 kDa. Elution of the latter band from SDS-PAGE demonstrated its capacity to induce EC and MP tissue factor activity, as well as to promote MP and PMN migration, as described previously (7) .
  • a series of peptides were prepared based on the N-terminal sequence of murine EMAP II (7) via solid phase methodology (22) using either t-boc or f-moc chemistry.
  • peptides were purified by HPLC and analyzed via mass spectrometry.
  • the peptide RIGRIIT was generously provided by Drs. Arun Patel and George Glover (SmithKline Beecha , King of Prussia PA) .
  • peptides were prepared with an additional C- terminal tyrosine to facilitate radioiodination by the chloramine T method (23) .
  • the final specific radioactivity of RIGRIVTAKY was 3xl0 5 cpm/ng.
  • Murine tumor necrosis factor- ⁇ (TNF) was purchased from Genzyme (Cambridge MA)
  • murine IL-l ⁇ (TNF) was generously provided by Dr. Peter Lomedico (Hoffmann- LaRoche, Nutley NJ)
  • formyl-methionyl-leucinyl- phenylalanine (fMLP) was obtained from Sigma (St. Louis MO) .
  • PMNs Preparation of PMNs. MPs. and ECs. Human PMNs were isolated from heparinized blood of normal volunteers by centrifugation over Histopaque 1119 (Sigma) . Pellets containing erythrocytes and PMNs were diluted 1:2 in normal saline, exposed to NaCl (0.2%) for 20 sec (to lyse erythrocytes) , restored to isotonicity, and centrifuged (350xg) for 10 min (7,24) .
  • the adherent cell population was harvested by incubation in calcium-magnesium-free buffer containing EDTA, as described previously (7,25), and MPs were resuspended in RPMI 1640 containing human serum (10%) at a density of 10 6 cells/ml.
  • Human umbilical vein ECs were prepared by the method of Jaffe (26) as modified by Thornton et al (27, and were characterized as described previously (28) . Experiments were carried out within 48 hrs of the cells achieving confluence.
  • Tissue factor activity of monolayers of ECs and MPs was determined (7) after incubation with EMAP II-derived peptides at 37°C for 4-12 hrs by washing cultures with
  • Factor Xa concentration was determined by comparison with a standard curve generated with known amounts of purified human Factor Xa. Human, plasma-derived Factors Vila and X were purified to homogeneity as described (31) .
  • Fluorescence of fura-2 was monitored at 37°C in a thermostatically-controlled cuvette installed in a Perkin Elmer Model 650-40 fluorescense spectrophotometer. Calibration of [Ca 2+ ]i was performed as described (34) .
  • PMNs myeloperoxidase
  • MTB peroxidase substrate 3,3' ,5,5' -tetramethylbenzidine
  • PMNs 3xl0 6 cells/ml; 0.05 ml
  • PMNs were incubated for 60 min at 37°C with RPMI 1640 containing fetal calf serum (1%) alone or in the presence of phorbol ester (phorbol 12-myristate 13-acetate; Sigma) or EMAP II- derived peptides.
  • Peroxidase activity assessed by oxidation of TMB, was determined spectrophotometrically and is reported as percent total peroxidase activity (100% is the activity observed with that number of PMNs following 60 min exposure to phorbol ester, 10 ⁇ M) .
  • a standard curve was generated by assaying peroxidase activity from different numbers of PMNs treated with phorbol ester (10 ⁇ M) under these conditions, and peroxidase activity of PMN/EMAP II peptide incubation mixtures was determined by comparison with the linear portion of the standard curve (35) .
  • Radioligand binding studies were performed on human MPs plated in 96-well plates (5-6 x 10 4 cells/well) . Cells were washed twice with Hanks balanced salt solution, and then Dulbecco's Modified Eagle Medium containing HEPES (25 mM; pH 7.4) , penicillin/streptomycin (50 U/ml; 50 ⁇ g/ml) , and bovine serum albumin-fatty acid free (0.5%; Sigma) were added (0.1 ml/well) . Cultures were incubated at 4°C for 2 hrs with 125 I-RIGRIVTAKY alone or in the presence of unlabelled peptide/protein.
  • myosin 200 kDa
  • phosphorylase b 97.4 kDa
  • bovine serum albumin 69 kDa
  • ovalbumin 46 kDa
  • carbonic anhydrase (30 kDa) trypsin inhibitor (21.5 kDa)
  • lysozyme (14.3 kDa).
  • Peptide-albumin conjugates made with either ASRLDLRIGRIVTAKY, RIGRIVTAKY or CRAQTMANSGIK
  • glutaraldehyde as described (39) .
  • mouse serum albumin 50 ⁇ g was incubated with glutaraldehyde (450 ⁇ g) and the indicated peptide (200 ⁇ g) in NaCl (0.1 M) Tris (0.05 M; pH 7.3) for 10 min at room temperature.
  • Excess lysine was added (final concentration, 0.5 M) , and the albumin-peptide conjugates were dialyzed exhaustively versus phosphate-buffered saline.
  • mice each received an injection into the footpad (7,40-41) of 0.05 ml of a solution of either (i) albumin-peptide conjugate (50 ⁇ g, total protein/footpad) , (ii) albumin treated with glutaraldehyde in an identical fashion, except that no peptide was present (50 ⁇ g, total protein/footpad) , (iii) native albumin (50 ⁇ g total protein/footpad) , or (iv) buffer alone.
  • animals were sacrificed by humane euthanasia, footpads were harvested, fixed in buffered formalin (10%) , decalcified, and embedded in paraffin. Sections were stained with hematoxylin and eosin.
  • ASRLDLRIGRIVTAKY concentration of -150-300 pM.
  • the peptide induced directional PMN migration rather than simply chemokinesis since addition of peptide to the upper well attenuated/abolished the response to ASRLDLRIGRIVTAKY added to the lower well.
  • ASRLDLRIGRIVTAKY also released PMNmyeloperoxidase activity in the peroxidase generation assay, as did phorbol ester-treated positive controls.
  • #Residues of peptides were assigned numbers (referred to in the text) starting with #6, N-terminal A, to #21, C-terminal Y. These numbers were based on the N- terminal protein sequence of EMAP II in which A was residue #6. *Cys at this position was carboxymethylated. For these studies, comparable molar concentration of peptide were employed, and where the data is reported as (+) , there was a similar response (the designation [-] indicated no response above that observed in untreated control wells) .
  • ASRLDLRIGRIVTAKY was found to induce chemotaxis, whereas the C-terminal EMAP II-derived peptide, as well as the IL-8-derived peptide and growth hormone-derived peptide were inactive, i.e., comparable to medium alone.
  • Migration of MPs in the presence of ASRLDLRIGRIVTAKY at a concentration of 100 pM was similar to that observed with FMLP at 1 ⁇ M.
  • EMAP II stimulated tissue factor expression in ECs and MPs (7) .
  • EMAP II-derived N-terminal peptide increases .Ca 2 * " !; in MPs and PMNs.
  • ASRLDLRIGRIVTAKY induced a rise in tCa 2+ ]i in PMNs, similar to results using the intact EMAP II molecule.
  • the rise in [Ca 2+ ]j was due mainly to redistribution of Ca + from intracellular stores since a similar increase was seen when the cells were incubated in Ca 2+ -free medium containing 5 mM EGTA.
  • mice Implantation of EMAPII-derived peptide-albumin conjugates into mice.
  • experiments were performed in vivo to determine if the peptide had the ability to incite an inflammatory response.
  • the mouse footpad was selected as a model system for these experiments since it is well- characterized and provides a relatively confined space for testing the host response to inflammatory cytokines, such as intact EMAP II as will as other mediators (7,40-41).
  • Initial experiments employing ASRLDLRIGRIVTAKY injected subcutaneously into mice demonstrated at most a transient inflammatory response, probably due to rapid diffusion of the small peptide away from the implantation site.
  • ASRLDLRIGRIVTAKY and RIGRIVTAKY were conjugated to albumin using glutaraldehyde, and the experiments were repeated.
  • ASRLDLRIGRIVTAKY-albumin induced both MP and PMN migration compared with medium alone. In contract, neither albumin treated with glutaraldehyde nor albumin alone induced migration.
  • RIGRIVTAKY-albumin conjugates were shown to have chemoattractant properties for PMNs and MPs, whereas CRAQTMANSGIK-albumin did not.
  • EMAP II a novel polypeptide mediator made by the immunogenic murine meth A fibrosarcoma, modulates cellular properties resulting in induction of tissue factor in ECs, in tissue factor and cell migration in MPs, and in cell migration and release of myeloperoxidase in PMNs (7 and unpublished observation) .
  • EMAP II a novel polypeptide mediator made by the immunogenic murine meth A fibrosarcoma, modulates cellular properties resulting in induction of tissue factor in ECs, in tissue factor and cell migration in MPs, and in cell migration and release of myeloperoxidase in PMNs (7 and unpublished observation) .
  • EMAP II a novel polypeptide mediator made by the immunogenic murine meth A fibrosarcoma
  • EMAP II cellular recognition site for the N- terminal region of EMAP II wa3
  • cytokine such as IL-1 or TNF
  • chemokine IL- 8 and related murine homologs
  • Meth A cell RNA Isolation of Meth A cell RNA.
  • Meth A cells were grown in RPMI 1640 containing fetal bovine serum (10%; Hyclone, Sterile Systems, Logan, UT) to -90% confluence, cells were harvested (-10 8 ) with trypsin, resuspended in fetal bovine serum (10%) , and poly(A) + RNA isolated directly as described (Bradley 1988) . Briefly, cells were lysed in SDS-containing buffer and proteins were digested with proteinase K
  • Oligo (dt) cellulose (Collaborative Biomedical,Bedford,MA) was added and the poly(A) + RNA removed by centrifugation and then eluted with water.
  • a second step in the purification utilized oligo(dt) cellulose bound to magnetic beads (Promega, locationx) by a similar procedure.
  • the thermocyte parameters consisted of three cycles of 95°C for 30 sec, one min to reach 37°C, 30 sec at 37°C, 2.5 min to 72°C, one min at 72°C, and one min to reach 95°. This was followed by 30 cycles of 30 sec at 95°C, 30 sec at 55°C, and one min at 72°C.
  • Analysis of the amplified products on an acrylamide gel showed a DNA fragment of the expected size of 77bp.
  • the PCR products were then digested with EcoRI, run on an acrylamide gel, the appropriate band excised and eluted, and the DNA fragment cloned into the plasmid vector pUC219.
  • Plasmids containing EcoRI inserts were sequenced by the Sanger dideoxynucleotide method using Sequenase (US Biochmical Corp.). The deduced amino acid sequence was found to match exactly that obtained by protein sequencing. A 57-mer nucleotide probe was designed based on the consensus nucleotide sequence obtained from sequencing several clones, as follows: ( 5 ' -
  • This probe was end-labelled with [r 32 P]dCTP using polynucleotide kinase and employed to screen a Meth A cDNA library in the lambda vector HEB05 (Leung et al, 1992). Hybridization was in formamide (20%), SSC (5x) , sodium phosphate (50 mM; pH 6.5), denatured salmon sperm DNA (40 ⁇ g/ml) , SDS (0.1%) , and Denhardt's solution (5x) at 42°C. One positive plaque was identified which contained a 700 bp insert.
  • This library was screened with the same oligonucleotide probe described above under the same conditions. Eight positive plaques were obtained from -10 5 screened. The three with the longest inserts, all -300 bp, were subcloned into the EcoRI site of pUC219 and sequenced. When this sequence was overlapped with the original clone, an 1086 bp sequence was obtained.
  • a full length EMAPII cDNA was constructed in the Epstein-Barr virus-based vector, pHEB023 (Leung et al, 1992) , by joining the two fragments at the Xbal restriction site present in both pieces.
  • a 279 bp DNA fragment was isolated from the murine EMAPII clone following Xbal and SacI digestion corresponding to nucleotides 652- 930. This was nick-translated with [o ⁇ 2 P]dCTP and hybridized to the blot overnight. Washing was performed at a final stringency of SSC (0.2x)/SDS (0.1%) at 55°C. The blot was then exposed overnight for autoradiography.
  • E. coli expression of murine EMAPII In order to confirm the biological activity of the protein encoded by the cloned DNA sequence, the region corresponding to the predicted mature protein, based on the N-terminal sequence obtained from purified EMAPII, was expressed. This was accomplished using a fragment of the murine clone extending from the BstB I site (nucleotide 529) to the 3' -untranslated region and synthetic DNA encoding the N- erminal end, KPIDASRLEL (5'TATGAAACCAATCGATGCATCTCGTCTGGATCTT-3' AND 5' - CGAAGATCC ⁇ GACGAGATGC ⁇ TCGATTGGTTTCA-3') .
  • Thissequence which differs from the amino terminal region obtained by microsequencing because the N-terminal residue, serine, was inadvertently omitted when designing the synthetic DNA, was cloned into the Ndel site, containing the ATG initiation codon, and the BamHI site of the vector pET-3a in which cDNA expression is driven by the T7 promoter (novagen) .
  • the protein was then expressed in the host HMS174(DE3) which contains the T7 RNA polymerase gene under control of the lacUV5 promoter. Following growth to log phase the T7 polymerase was induced with IPTG (0.4 mM) and the cells were harvested by centrifugation 3 hr later.
  • Meth A cells a methylcholanthrene A-induced fibrosarcoma originally derived from BALB/c mice were generously provided by Dr. L. Old (Ludwig Cancer Inst., NY) .
  • Cells were grown in RPMI 1640 (Gibco BRL, Grand Island, NY) containing fetal calf serum (10%; HyClone Laboratories, Logan, UT) penicillin/streptomycin (lx; Gibco BRL) , and L- glutamine (2mM) (this is termed complete medium-CRPMI) , and maintained at 37°C in a humidified, 5% C0 2 atmosphere.
  • the medium for selection of transfectants contained G418 (400 ⁇ g/ml; Gibco BRL) .
  • Full-length EMAP II cDNA was subcloned into the pRK5 plasmid.
  • Exponentially growing Meth A cells were transfected with Cel-Porator electroporation System I (Gibo BRL) . Briefly, serum-free RPMI (1 ml) containing meth A cells (2x10°), pRK5-proEMAPII cDNA (20 ⁇ g) , and pRK5-Neo DNA (1 ⁇ g; containing the G418 resistence gene) was transferred into the electorporation chamber, and the electorporation ws conducted at 250 V.
  • the cells were incubated for 15 min at 23°C, and then transferred to CRPMI (15 ml) . After a further 24 hrs, cells were pelleted, resuspended in the selective medium containing G418 (400 ⁇ g/ml) , and aliquoted into four 48-well plates. Cells were re-fed with selective medium every three days, and 2-3 weeks later colonies became visible. Wells with a single colony were chosen for expansion. Production of EMAPII antigen was quantitated by ELISA.
  • PMNs polymorphonuclear leukocytes
  • Human umbilical vein ECs were prepared by the method of Jaffee et al as modified by Thornton et al, as done previously (7) .
  • Bovine aortic ECs were harvested fron the aortae of veal calves and grown in culture as described previously.
  • Human peripheral blood monocytes were isloated from the blood of normal healthy volunteers.
  • RESULTS cDNA cloning of EMAPII Purification of EMAPII from conditioned medium of murine meth A sarcoma yielded an -22 kDa protein from which an unique amino terminal sequence was obtained. Degenerate oligonucleotide primers were designed to generate a 77bp fragment encoding a portion of the N-terminal sequence by PCR. The sequence obtained was then used as the basis for design of a 57 base oligonucleotide probe for screening a meth A oligo(dt) -primed cDNA library. One clone, 680 bp, was isolated and represented a partial cDNA sequence with an open reading frame at the 5' -end.
  • a second meth A cDNA library was constructed using a specific primer based on the first sequence obtained, and the same 57 base probe was used to identify 8 clones. Three of these appeard to have identical inserts of 660 bp based on restriction analysis, and the sequence obtained from these clones was overlapped with that of the original clone to produce contiguous sequence of 1086 bp (Fig. 4) .
  • Northern blot analysis of RNA from Meth A cells using a 275 bp Xbal to Sad fragment as a probe demonstrated a single transcript of approximately 1070 bp suggesting the cDNA clone was full-length. Analysis of this sequence revealed an open reading frame containing residues encoding the N- terminal sequence with a termination codon at the nucleotide 894 followed by a polyadenylation signal
  • AATAAA AATAAA
  • codon 64 the open reading frame would encode a protein of 310 amino acides with a predicted molecular wieght of -34 kDa.
  • a fragment of the murine cDNA was used as a probe to isolate a full-length human cDNA clone for EMAPII from a lambda gtll U937 monocyte library (Fig. 4) .
  • the human cDNA was 86% identical, and the deduced sequence contained an additional two amino acids.
  • the ATG designated as the start codon and the upstream ATG are both conserved in the human cDNA.
  • the N-terminal sequence obtained from the purified EMAPII is encoded by an internal sequence of the EMAPII clone, it was predicted that mature EMAPII results from processing from a larger polypeptide.
  • the cDNA corresponding to the N-terminal, processed portion of the sequence encodes 165 amino acids which would result in a polypeptide of -18 kDa, in close agreement with the -22 kDa observed for EMAPII purified from meth A sarcoma cells.
  • EMAPII is apparently secreted by meth A sarcoma cells, a hydropathy analysis of the predicted murine primary amino acid sequence lacked evidence for a hydrophobic signal peptide.
  • IL-10 interleukin-10
  • the mRNA for IL-ljS encodes a 31 kDA precursor to the mature 17 kDA from (March et al, 1985) , and proteolytic processing releases a 17 kDa secreted, active IL-l/S (Black, 1989) .
  • a cystine protease similar or identical to ICE might be responsible for producing mature EMAPII from its pro-from.
  • sequence conservation is 95% between the murine and the human region of the mature polypeptide, but drops to 74% in the putatuive pro-region.
  • EMAPII The primary amino acid sequence of EMAPII shows little homology to any other proteins in the data banks. Nevertheless, a limited resemblance exists between residues in the N-terminal portion of EMAPII and several other cytokines, notably IL-8 and IL-lS, as well as von Willebrand antigen II, a product released by activated platelets and endothelial cells. All of these molecules share chemoattractant properties towards neutrophils and/or monocytes (Yoshimira, 1987; Sauder, (18)).
  • E. coli transfected with the portion of the EMAPII cDNA corresponding to mature EMAPII were pelleted by centrifugation, sonicated in the presence of tris- buffered saline, and the supernatants chromatographed on FPLC Mono Q.
  • the peak containing EMAPII activity was identified based on the induction of tissue factor activity in ECs. In contrast, little protein eluted at a similar salt concentration from material obtained from E. coli transformed with vector alone, and this small peak had no significant tissue factor inducting activity.
  • the material from the Mono Q activity peak of the E was selected from the Mono Q activity peak of the E.
  • mice Normal C3H/He mice were injected intradermally with 20 micrograms of EMAP II. Each mouse received 100 micrograms of endotoxin, systemically, either 9, 15, or
  • mice Localized Thrombohemorrhage Normal Balb/C mice were injected intradermally with 20 micrograms EMAP II. Each mouse received 100 micrograms of endotoxin, systemically, either 18 or 24 hours after the EMAP II. Skin was harvested three hours later. In each case, localized hemorrhage was observed in the skin, at the site of the initial EMAP II injection.
  • mice were raised in the backs of C3H/He mice by intradermal injection of 2xl0 5 tumor cells. Seven days later, mice were given a single intratumor injection of either purified recombinant human Tumor Necrosis Factor (TNF, 5 micrograms, in a PBS/albumin vehicle) , heat treated TNF (inactivated by 15 minutes in a boiling water bath) , EMAP II (20 micrograms in the vehicle) , heat- treated EMAP II, or vehicle solution alone.
  • TNF Tumor Necrosis Factor
  • mice Six hours after injection, mice were sacrificed and tumors were observed for the presence of gross hemorrhage. EMAP II elicited gross hemorrhage in a proportion of tumors comparable to TNF, but the controls were without appreciable effect. (Fig. 5) .
  • Experiment 4 Hemorrhage in Mouse Mammary Carcinomas (Single Injections)
  • mice Mouse mammary carcinomas derived from MC2 cell line were raised in the backs of C3H/He mice by intradermal injection of 10 6 tumor cells. Seven days later, mice were given a single intratumor injection of 5 micrograms TNF, heat-treated TNF, 20 micrograms EMAP II, heat-treated EMAP II, or vehicle alone. Six hours later, mice were sacrificed and tumors were observed for the presence of gross hemorrhage. No treatment elicited hemmorhage above baseline. (Fig. 6) .
  • mice Hemorrhage in Mouse Mammary Carcinomas (Dual Injections)
  • Mouse mammary carcinomas derived from the MC2 cell line were raised in the backs of C3H/He mice by intradermal injection of 10° tumor cells.
  • mice received intratumor injections of 20 micrograms EMAP II followed 12-18 hours later by a systemic dose of 5 micrograms TNF.
  • Control animals received combinations of either heat-treated (H.T.) EMAP II + TNF, EMAP II +
  • Mouse mammary carcinomas were treated as in experiment 5 with a local injection of 20 micrograms EMAP II followed 12-18 hours later by a systemic dose of 5 micrograms TNF, with control animals receiving heat- treated cytokines or vehicle. Length, width and height of each tumor was measured prior to the systemic dose and again on days 1, 3, and 7 after the systemic injection. Tumor volume was calculated by assuming the shape of each tumor was that of a spherical segment, and according to the formula:
  • V (pi/6)h(h 2 +3a 2 ),
  • EMAP II + TNF treatment is compared with each combination of heat-inactivated control, as well as to vehicle + TNF. (Fig. 8A-E) .
  • Silverstein S. (1988) J. Cell Biol. 106, 657-666. 34. DiVirgilio, F. , Steinberg, T. , and Silverstein, S.
  • Endothelial-monocyte activating polypeptide II was initially identified in the supernatant of murinemethylcholanthrene A-induced fibrosarcomas (Meth A) by its capacity to activate host effector cells )Kao, J., Ryan, J., Brett, J., Chen, J., Shen, H., Fan, Y-G., Godman, G., Familletti, P., Wand, F., Pan, Y-C, Stern, D., and Clauss, M. (1992) J. Biol. Chem 267, 20239-20247) .
  • EMAP II is a unique, leaderless, single polypeptide chain with predicted molecular mass -34 kDa and that the mature form released by Meth A cells corresponds to -20 kDa.
  • Purified recombinant mature EMAP II (EMAP II, -20 kDa form) activated endothelial cells with resulting elevation of cytosolic free calcium concentration, release of von Willebrand factor, induction of tissue factor, and expression of the adhesion molecules E- selectin and P-selectin.
  • EMAP II Neutrophils exposed to EMAP II demonstrated elevated cytosolic free calcium concentration, peroxidase generation, and chemotaxis. EMAP II also activated mononuclear phagocytes elevating cytosolic free calcium concentration, inducing tumor necrosis factor- ⁇ (TNF) and tissue factor, and stimulating chemotaxis. Systemic infusion of EMAP II into C3H/HeJ or Balb/c mice was associated with systemic toxicity, pulmonary congestion, and the appearance of TNF, interleukin-1 and -6 in the plasma. A single intra-tumor injection of EMAP II into Meth A sarcomas induced acute thrombohemorrhage and partial tumor regression.
  • TNF tumor necrosis factor- ⁇
  • EMAP II a tumor-derived cytokine
  • Tumor vasculature represents a critical link between the host and neoplasm. It serves as a conduit for the delivery of nutrients necessary to sustain the tumor and promote its growth and spread, but it also serves as a portal for the entry of host effector cells and cytotoxic agents (1,2) .
  • the vasculature of certain tumors has properties which distinguish it from the normal vasculature; these include an exaggerated response to the systemic infusion of flavone acetic acid or cytokines, such as interleukin 1 (IL-1) 1 and. tumor necrosis factor- ⁇ ; (TNF) , increased permeability, and infiltration by inflammatory/immune effector cell
  • Method A A-induced fibrosarcoma (Meth A) have been characterized based on their capacity to modulate properties of endothelial cells (ECs) , mononuclear phagocytes (MPs) , and polymorphonuclear leukocytes (PMNs) , cells which have important roles in tumor vasculature (11-13) .
  • ECs endothelial cells
  • MPs mononuclear phagocytes
  • PMNs polymorphonuclear leukocytes
  • Meth A tumors provide a suitable model for assessing the effects of tumor-derived factors which modulate the host response (4, 14) .
  • the vasculature of these tumors is distinguished from that of normal tissues by the properties described above.
  • the response of Meth A tumors to systematically administered cytokines has been well characterized, beginning with hemorrhage/thrombosis and increased permeability of tumor vessels, and associated with diminished blood flow as well as subsequent influx of hose effector cells (4) .
  • Meth A cells produce multiple mediators capable of interacting with inflammatory/immune effector cells, three polypeptides which are likely to contribute to host-tumor interactions have been characterized in more detail (11-13) .
  • EMAPs endothelial-monocyte activating polypeptides
  • RNA - Meth A cells (generously provided by Dr. L. Old, Cancer Research Institute, New York, NY; 14) were grown in RPMI 1640 containing fetal bovine serum (10%; Gemini, Calabsssas, CA) to -90% confluence. Cells (—10 s ) were harvested with trypsin, resuspended in fetal bovine serum (10%), and poly(A)+ RNA isolated directly as described (16) .
  • oligo(dT) 17 was used as a primer.
  • the first strand cDNA obtained was used as template for the polymerase chain reaction (PCR) using degenerated primers based on the NHj-terminal protein sequence obtained for EMSP II (13) .
  • the sense primer was 5' -GGCGAATTCA7RCCNATHGAYGC-3' and the antisense primer was 5' -GGCGAATTCYTTNGCNGTNACDAT-3' with EcoRI sites near their 5' -ends to facilitate cloning of the PCR products.
  • thermocycling parameters consisted of three cycles of 95°C for 30 s, 30 s at 37 reputationC, and 1 min at 72°C This was followed by 30 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C Analysis of the amplified products on an acrylamide gel showed a DNA fragment of the expected size of 77 bp.
  • the PCR products were then digested with EcoRI, run on an acrylamide gel, the appropriate band excised and eluted, and the DNA fragment cloned into the plasmid vector pUC219. Plasmids containing EcoRI inserts were sequenced by the Sanger dideoxynucleotide method using Sequenase (U.S.
  • This probe was end-labeled with 32 P-dCTP using polynucleotide kinase and employed to screen a Meth A cDNA library in the ⁇ vector HEB05 (17) .
  • Hybridization was performed in formamide (20%) , SSC (5 x) , sodium phosphate (50 mM, pH 6.5) , denatured salmon sperm DNA (40 ug/ml), SDS (0.1%), and Denhardt's solution (5 x) at 42°C
  • One positive plaque was identified which contained a 700-bp insert.
  • a second library in ⁇ gt 10, was constructed from cDNA primed with a specific primer, 5' -ATTTTGCATCTGTTCTAG-3' , complementary to sequence near the 5' -end of the original clone.
  • This library was screened with the same oligonucleotide probe described above under the same conditions.
  • Eight positive plaques were obtained from -10 5 screened. These were sub-cloned in pUC219 and sequenced in both directions. When this sequence was overlapped with the original clone, a 1086-bp sequence was obtained.
  • a full-length EMAP II cDNA was constructed in the Epstein-Barr virus-based vector, pHEB023 (17) , by joining the two fragments at the Xbal restriction site present in both pieces.
  • This Clal (in the vector polylinker) to Seal (500 bp) fragment was nick- translated and hybridized in formamide (20%) , sodium phosphate (50 mM, pH 6.5), SSC (5 x) , Denhardt's solution (5 x) , SDS (0.1%), and denatured salmon sperm DNA (40 ug/ml) at 42°C About 20 positives were obtained, and 10 of these were purified. The three which contained the longest inserts appeared to have identical 1100-bp EcoRI inserts. These inserts were subcloned in pUC219 and sequenced.
  • RNA was transferred to nitrocellulose (Schleicher and Schuell) and prehybridized in formamide (50%) , sodium phosphate (50mM, pH 6.5), SSC (5 x) , DeDenhardt's solution (5 x) , SDS (0.1%), and denatured salmon sperm DNA (40 ug/ml) at 42°C
  • formamide 50%
  • sodium phosphate 50mM, pH 6.5
  • SSC 5 x
  • DeDenhardt's solution 5 x
  • SDS 0.1%)
  • denatured salmon sperm DNA 40 ug/ml
  • E. coli Expression of Murine EMAP II - In order to confirm the biological activity of the protein encoded by the cloned DNA sequence, the region corresponding to the predicted mature protein, based on the NH 2 -terminal sequence obtained from purified EMAP II, was expressed. This was accomplished using a fragment of the murine clone extending from the BstBI site (nucleotide 529) to the 3' -untranslated region and synthetic DNA encoding the NH 2 -terminal end, KPIDA-SRLEL (5'- TATGAAACCAATCGATGCATCTCGTCTGGATCTT-3' and 5' - CGAAGATCCAGACGAGATGCATCGATTGGTTTCA-3' ) .
  • Thissequence which differs from the NHj-terminal region obtained by micro-sequencing because the NH 2 -terminal residue, serine, was inadvertently omitted when designing the synthetic DNA, was cloned into the Ndel site, containing the ATG initiation codon, and the BamHI site of the vector pET-3a in which cDNA expression is driven by the T7 promoter.
  • the protein was then expressed in the host HMS174(DE3) which contains the T7 RNA polymerase gene under control of the lacUV5 promoter. Following growth to log phase, the T7 polymerase was inducedwithisopropyl-1-thio-?-D-galactopyrnoside (0.4 mM) , and the cells were harvested by centrifugation 3 h later.
  • the pellet was dissolved in Tris (20 mM, pH 7.4)/EDTA (2 mM)/benzamidine (1 mM)/sodium azide (0.02%)/octyl-B- glucoside (0.1%), agitated at 4°C, and sonicated. The supernatant was centrifuged at 20,000 revolutions/min for 40 min (4°C) , and dialyzed versus Tris (20 mM, pH 7.4)/octyl-B-glucoside (0.1%). After dialysis, the sample (30-40 mg) was applied to FPLC Mono Q (HR 5/5) , and the column was eluted with an ascending salt gradient (0-1.0 M) . Fractions were assayed for their ability to induce EC tissue factor (see below) , and active material was pooled, concentrated (Centricon 3,
  • Characterization of purified recombinant EMAP II included SDS-PAGE, with visualization of protein by silver staining, and immunoblotting with an antibody generated to a synthetic peptide comprising residues 1- 14 from mature EMAP II (residues 145-158 from the precursor form) (13) . Sites of binding of primary antibody were visualized with peroxidase-conjugated goat anti-rabbit IgG (Sigma) .
  • Human peripheral blood monocytes were isolated from the blood of normal healthy volunteers (22) . Blood was centrifuged on Histopaque 1077 (Sigma) , the mononuclear fraction was obtained, washed twice in Hanks' balanced salt solution, resuspended in RPMI 1640 containing human serum (10%; Gemini, Calabassas CA) , plated on tissue culture dishes (106 cells/ml) , and incubated at 37°C for 1-2 h.
  • Nonadherent cells were removed by washing the plate three times with balanced salt solution, and adherent cells were harvested by incubation with calcium-magnesium-free buffer containing EDTA (2mM) for 15 min at 37°C, followed by extensive washing.
  • the adherent population which is enriched for human mononuclear phagocytes (although there are also some additional cells) were termed MPs.
  • Cytosolic [Ca 2+ ] and chemotaxis experiments employed freshly isolated MPs. For other experiments, MPs were allowed to differentiate in culture for 10-14 days is RPMI 1640 containing human serum (10%) and penicillin/streptomycin (100 units/ml-100 ug/ml) .
  • Human PMNs were prepared from heparinized blood by centrifugation over Histopaque 1077 and 1119 (Sigma) .
  • Cell pellets containing red cells and PMSs were diluted 1:2 in normal saline, exposed to NaCl (0.2) for 20 s (to lyse erythrocytes) , restored to isotonicity with sodium chloride (1.6%), and centrifuged (250 x g) for 10 min (13, 23). Following two repetitions of this procedure, the cell population of .98% PMNs was resuspended in RPMI 1640 containing heat-inactivated human serum (3%) .
  • PCR analysis to detect tissue factor transcripts was performed on human mononuclear phagocytes and ECs as described previously (13) .
  • Controls included amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts (13) .
  • GPDH glyceraldehyde phosphate dehydrogenase
  • Ec expression of the leukocyte adherence molecule E- selectin was monitored by ELISA (R&D Systems, Cambridge, MA) on the surface of nonpermeabilized, fixed ECs, as described (26) .
  • ECs were exposed to medium alone, LPS or EMAP II. for 4 h, cultures were then fixed in glutaraldehyde (0.05%) , blocked with bovine serum albumin (3%) , and incubated with mouse anti-human E-selectin IgG followed by peroxidase-conjugated goat anti-mouse IgG.
  • Western blotting was performed on EC monolayers exposed to the above stimuli by dissolving the cells in lysis buffer
  • ECs were incubated with either EMAP II or a-thrombin (2 units) for 1 h, foxed for 15 min in paraformaldehyde (1%; 29), and exposed to 15 I-anti-P-selectin IgG (100 ng) for 2 h at 4°C Cultures were then washed four times in balanced salt solution, and bound antibody was eluted by dissolving the cell monolayer with Triton X-100 (1%) in phosphate-buffered alsine. Nonspecific binding of 125 I- anti-P-selectin antibody to EC monolayers was determined in the presence of a 1,000-fold excess of unlabeled anti-P-selectin antibody.
  • Specific binding is the difference of total binding (in the presence of 125 I-anti-P-selectin IgG alone) and nonspecific binding (in the presence of excess unlabeled anti-P-selectin IgG) .
  • Data in Fig. 7C are reported as a percent of the specific 125 I-anti-P-selectin IgG binding observed following exposure of monolayers to thrombin (the positive control) .
  • EC release of vWF into culture supernatants was quantified by ELISA.
  • the stimulus was placed in the upper or lower chamber, as indicated, and the cells were allowed to migrate for 3 h (for MPs) or 45 min (for PMNs) at 37°C Following removal of nonmigrating cells, membranes were fixed in methanol, and migrating cells were visualized with Wright's stain. Assays were performed in quadruplicate, and cells were counted in eight high- power fields in each case (mean ⁇ S.E. is shown in the figures) .
  • TNF Production by Human Macrophages - Production in RPMI containing heat-treated human serum (1%) was studied by incubating cultures (10 5 cells/well) with EMAP II for 6 h and assaying aliquots of culture supernatant using an ELISA for TNF ⁇ antigen.
  • PCR analysis for human "TNF ⁇ . transcripts was also performed on cultured macrohpages exposed to EMAP II by extracting total RNA using the acid-guanidinium thiocyanate procedure (Stratagene Inc., Torrey Pines, CA) . Random hexanucleotide-primed first strand cDNA was prepared and served as template for PCR analysis. TNF primers were those described previously (38) .
  • cDNA was amplified by PCR for 30 cycles (with TNF primers) and 20 cycles (with GAPDH primers) , each cycle consisting of incubations at 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min. Products were analyzed by agarose gel electrophoresis (1%) and visualized by ethidium bromide staining under ultraviolet light.
  • IL-8 production by human MPs in RPMI 1640 containing heat-treated human serum (1%) was studied by exposing cultures (105 cells/ well) to EMAP II for 8 h and assaying aliquots of culture supernatant in an ELISA for human IL-8 as reported (39) .
  • EMAP II-induced changes in IL-8 transcripts were studied by PCR, as described above.
  • IL-8 primers were those described by Carre et al . (40) and GAPDH primers used were as above. Conditions for PCR consisted of incubations at 95°C for 2 min for 1 cycle, 1 min at 95°C followed by 1 min at 60°C for 35 cycles, and 7 min at 60°C for 1 cycle for IL-8 and GAPDH.
  • mice Female mammary carcinomas derived from the MC2 cell line (generously provided by Dr. Jan Vaage, Rosewell Park Cancer Institute, Buffalo, NY) (44) were raised by intradermal injection of 106 tumor cells into the dorsal skin of female C3H/He mice.
  • each mouse received an intratumor injection of EMAP II (10 ug in vehicle) , heat-treated EMAP II (10 ug) , or vehicle alone (same volume as other injections) , followed 12-18 h later by a single tail vein injection of either TNF (5 ug) , heat-treated TNF (5 ug) , EMAP II (20 ug) , or heat-treated EMAP II (20 ug) .
  • Tumors were either excised 6 h following the intravenous injection and examined for the presence of hemorrhage, or else followed for up to 7 days for assessment of tumor volume (as above) over time.
  • groups of three tumors were pooled and assayed for clonogenic cell survival by a modification of the methods of Twentyman et al . (45) and Braunschweiger et al . (46) .
  • Tumors were aseptically excised and weighed, then finely minced, and digested by gently agitation for 90 min at room temperature in Hanks' balanced salt solution containing trypsin (0.75 mg/ml), collagenase (0.75 mg/ml) , and DNase (0.05 mg/ml) (all from Sigma) .
  • the resulting cell suspension was passed thro ⁇ gh a sterile wire mesh filter (Collector tissue sieve, Bellco Glass, Inc., Vineland, NJ) , washed twice, counted, and plated out at serial dilutions in RPMI 1640 containing fetal calf serum (10%) , penicillin (150 units/ml) , and streptomycin (150 ug/ml) . After 4 days, the number of dividing colonies (each reflecting an initially clonogenic cell) derived from treated and control tumors were counted by a blinded observer. The proportion of clonogenic cells/gram of tumor, relative to control, was taken as the surviving fraction. All experiments involving tumor growth were performed with age- and sex-matched controls.
  • EMAP II EMAP II from conditioned medium of murine Meth A sarcoma cells yielded a -22-kDa protein from which a unique amino-terminal sequence was obtained (13) .
  • Degenerate oligonucleotide primers were designed to generate a 77-bp fragment encoding a portion of the NH2-terminal sequence by PCR. The sequence obtained was then used as the bases for design of a 57-base oligonucleotide probe for screening a Meth A oligo(dT) -primed cDNA library.
  • a second Meth A cDNA library was constructed using a specific primer based on the first sequence obtained, and the same 57-base probe was used to identify eight clones. Three of these appeared to have identical inserts of 660 bp based on restriction analysis, and the sequence obtained from these clones was overlapped with that of the original clone to produce a contiguous sequence of 1086 bp (Fig. 4) .
  • Northern blot analysis of RNA from Meth A cells using a 275-bp Xbal to SacI fragment as a probe demonstrated a single transcript of -1070 bp suggesting the cDNA clone was full-length (Fig. 11) .
  • a fragment of the murine cDNA was used as a probe to isolate a full-length human cDNA clone for EMAP II from a ⁇ gt 11 U937 monocyte library (Fig. 13) .
  • the human cDNA was 86% identical, and the deduced sequence contained an additional 2 amino acids.
  • the ATG designated as the start codon and the upstream ATG are both conserved in the human cDNA.
  • NHj-terminal sequence obtained from purified EMAP II is encoded by an internal sequence of the EMAP II clone, it was predicted that mature EMAP II results from processing of a larger polypeptide.
  • the cDNA corresponding to the NH 2 -terminal, processed portion of the sequence encodes 165 amino acids which would result in a polypeptide of —18 kDa, in close agreement with the -22 kDa observed for EMAP II purified from Meth A sarcoma cells.
  • EMAP II is apparently secreted by Meth A sarcoma cells (13) , hydropathy analysis of the predicted murine primary amino acid sequence lacked evidence of a hydrophobic signal peptide (Fig. 12) .
  • IL-lS secreted cytokine which also lacks a classic signal peptide
  • the mRNA for IL-l/S encodes a 31-kDa precursor to the mature 17 kDa form (48) .
  • the pre-IL-1S is cell associated, unable to bind to IL-1 receptors, and is biologically inactive (49) .
  • Proteolytic processing releases a 17- kDa secreted, active IL-lS (50) .
  • EMAP II The primary amino acid sequence of EMAP II showed little homology to any other proteins in the data banks. Nevertheless, a limited resemblance existed between residues in the NH2-terminal portion of mature EMAP II and several other cytokines, notably IL-8 (54, 55) and IL-l/S (48) , as well as von Willebrand antigen II, a product released by activated platelets and endothelial cells (56-60) (Fig. 13). All of these molecules share chemoattractant properties toward neutrophils and/or monocytes (61-63) .
  • E. coli transformed with the portion of the EMAP II cDNA corresponding to mature EMAP II were pelleted by centrifugation, sonicated in the presence of Tris- buffered saline, and the supernatants chromatographed on FPLC Mono Q (Fig. 14A) .
  • the peak containing EMAP II activity was identified based on induction of tissue factor activity in ECs. In contrast, little protein eluted at a similar salt concentration from material obtained from E. coli transformed with vector alone, and this small peak had no significant tissue factor inducing activity when applied to ECs (Fig. 14b) . Active fractions eluted from the Mono Q column (Fig.
  • Rises in EC cytosolic calcium are more typically identified with mediators such as histamine and thrombin (65, 66) , rather than cytokine- like molecules such as EMAP II.
  • EMAP II also modulated EC functions by causing the expression of gene products not normally present in quiescent ECs.
  • Analogous to previous results with EMAP II purified from Meth A cells (13) recombinant EMAP II induced tissue factor activity in ECs in a dose- dependent manner, with half-maximal effect at —2-5 PM (Fig. 8A) , and in a time-dependent manner (Fig. 17B) .
  • Heat-treated EMAP II was inactive.
  • the necessity for de novo biosynthesis in EMAP II-induced expression of EC tissue factor was indicated by the inhibitory effect of actinomycin D (Fig. 17A) .
  • EMAP II increased tissue factor transcripts (Fig. 17C, tissue factor, TF) , whereas levels of transcripts for GAPDH remained unchanged (Fig. 17C GAPDH) .
  • E-selectir a cell surface ELISA for E-selectin demonstrated that ECs exposed to EMAP II expressed E-selectin antigen, comparable to levels observed following LPS stimulation, in contrast to undetectable levels in untreated controls (Fig. 17D) .
  • the presence of E- selectin antigen was confirmed by Western blotting of ECs exposed to EMAP II, where a single major band was observed (Fig. 17E, EMAP II) comigrating with that induced after exposing ECs to LPS (Fig. 17E, LPS) .
  • EMAP II Leukocytes (PMNs) - Because EMAP II could potentially increase adhesivity of PMNs for ECs by P-selectin- and E-selectin-mediated mechanisms, it was important to determine whether EMAP II would also directly modulate properties of leukocytes. EMAP II elevated [Ca 2+ ] , in
  • FIG. 18A since it was seen only in cells incubated in the presence of ImM extracellular Ca + (data not shown) .
  • EMAP II induced concentration-dependent increase in myeloperoxidase activity, as measured in the peroxidase generation assay. This occurred in a dose-dependent fashion (Fig. 18B; EMAP II, bars 4-6) and was comparable to peroxidase generation by phorbol- ester (Fig. 18B; PMA, bars 1 -3) .
  • Heat-treated EMAP II had the same effect on myeloperoxidase release as did medium alone (Fig. 18B; heat treatment (H. T. ) and medium, bars 7 and 8, respectively) .
  • Meth A-derived EMAP II Stimulates monocyte migration, and also shortens the clotting time of murine MPs (13) , suggesting induction of procoagulant activity, the effect of recombinant EMAP II on these cellular properties was examined.
  • Recombinant EMAP II induced directional migration of human MPs in a dose-dependent manner (Fig. 19E) , over a similar range of concentrations to that observed for inducing PMN migration (Fig. 18C) .
  • Recombinant EMAP II also induced
  • FIG. 19F which was accompanied by elevation of tissue factor transcripts (Fig. , 10G, TF) .
  • Heat treatment of EMAP II abrogated its ability to induce tissue factor activity in MPs (Fig. 19F) .
  • Tissue factor induction in MPs was prevented by addition of actinomycin D, and tissue factor activity in the Factor VIla-dependent Factor Xa formation assay was completely blocked by neutralizing anti-tissue factor IgG (data not shown) .
  • EMAP II would be phlogogenic in the intact animal, and indeed earlier studies in the mouse footpad showed that Meth A-derived EMAP II induced an inflammatory -Ill- response (13) .
  • tow models were selected: systemic infusion into normal mice and intratumor injection in mice bearing Meth A and murine mammary tumors.
  • lung tissue from treated animals demonstrated arrest of circulating leukocytes/inflammatory cells in the pulmonary vasculature compared with untreated controls (Fig. 20, C and D) .
  • Fig. 20, C and D As an index of pulmonary leukostasis, sequential myeloperoxidase activity was determined in lung tissue from mice infused with EMAP II: myeloperoxidase activity rose within 0.5 H, remaining elevated for about 3 h (Fig. 20B) . Animals receiving heat-inactivated EMAP II showed no similar increase in myeloperoxidase activity.
  • infusion of EMAP II produced a similar pattern of cytokine inductipn.
  • EMAP II Intratumor Injection of EMAP II -
  • the heterogeneity of the Meth A tumors, with areas of necrosis and infiltration by immune/inflammatory cells (4, 14) suggested the possibility that low concentrations of locally produced mediators, such as EMAP II, had perturbed the microenvironment.
  • administration of a single dose of EMAP II directly into the malignant lesion might result in changes including thrombohemorrhage and subsequent tumor regression.
  • C3H/He mice bearing Meth A tumors received a single intratumor injection of either EMAP II, heat-treated EMAP II, TNF, heat-treated TNF, or vehicle alone.
  • EMAP II endogenous production of EMAP II serves to shift the phenotype of tumor vascular endothelium to a surface promoting thrombohemorrhage and leukocyte adhesivity, enabling subsequent administration of systemic mediators to induce profound thrombohemorrhage and intense inflammation localized to the tumor bed. It was postulated that local EMAP II treatment might render TNF-insensitive tumors susceptible to the effects of TNF.
  • a murine mammary carcinoma (MC2) was selected for these studies (44) since it does not undergo thrombohemorrhage or regression in response to TNF 2 . Intratumor injection of either TNF or EMAP II alone did not induce visible hemorrhage in mammary tumors (Fig. 22A) .
  • EMAP II followed -15 h later by intravenous TNF showed increased intratumor hemorrhage 6 h later as compared with controls (i.e. animals that received substitutions of heat-inactived EMAP II, heat-inactivated TNF, or both, in place of the active cytokines; p ⁇ 0.005) (Fig. 22B) .
  • Microscopic examination of tumors from animals which received EMAP II followed by TNF revealed an acute inflammatory infiltrate composed primarily of PMNs associated with tumor cell lysis within 6 h compared with controls (Fig. 22, C and D) .
  • EMAP II A critical factor contributing to the properties of tumor neovasculature is the effect of mediators made by the tumor cells on elements of the vessel wall and circulating leukocytes and monocytes.
  • EMAP II The only significant region of EMAP II with homology to previously described mediators (IL-8 and IL-l/S) is a locus proximal to the NH2 terminus of mature EMAP II which is also similar to von Willebrand factor antigen II.
  • a peptide comprising this portion of EMAP II has recently been shown to modulate properties of MPNs and MPs and to induce an acute inflammatory response when injected into the mouse footpad (25) . Consistent with these data, the homologous peptide from von Willebrand antigen II effectively modulates these same properties in PMs and PMNs (62) .
  • EMAP II The phlogogenic potential of EMAP II was suggested by in vitro studies demonstrating activation of ECs, MPs, and PMNs by several criteria, including direct effects on cellular properties and release of proinflammatory cytokines, such as TNF ⁇ and IL-8
  • Administration of EMAP II to mice resulted in evidence of systemic toxicity, as well as release of cytokines into the plasma.
  • the pattern of cytokine elaboration following infusion of EMAP II was somewhat different than that observed with LPD (69) ; in the former, TNF ⁇ appeared later, while IL- 6 was detected earlier.
  • EMAP II also produced a more sustained elevation in plasma levels of IL-lS.
  • EMAP II has the potential to modulate tumor vasculature by promoting thrombohemorrhage and increasing vascular permeability
  • EMAP II EMAP II-induced compromise of tumor vasculature.
  • Injection of EMAP II into a tumor which is capable of eliciting an inflammatory response, such as the Meth A sarcoma (4) augments tumor thrombohemorrhage, acute inflammation in the tumor bed, and subsequent tumor regression.
  • Administration of EMAP II to a tumor which is not initially sensitive to TNF, such as the MC2 murine mammary carcinoma, resulted in a local priming effect permitting subsequent thrombohemorrhage and regression once the tumors were treated with systemic TNF or EMAP II.
  • EMAP II-induced TNF production at the site of the tumor would be sufficient to initiate thrombohemorrhage
  • the priming dose of EMAP II had to be followed by a provocative challenge with TNF for the tumor vascular response to be observed.
  • the potential of locally injected EMAP II to prepare the site for a later severe inflammatory response is reminiscent of the preparatory injection in the Shwartzman reaction (70, 71) and suggests its potential in the therapy of tumors which are relatively resistant to cytokines such an TNF.
  • recent pilot studies suggest that EMAP II similarly primes the tumor bed of B16 melanomas and human fibrosarcomas (the later grown in immunodeficient mice) to undergo an acute inflammatory response followed by tumor regression.
  • EMAP II results in the tumor bed. Based on the protracted duration of inflammation following local injection of EMAP II into the tumor bed, compared with a much briefer acute inflammatory response following injection of EMAP II into normal tissue (13; also, EMAP II-induced changes in properties in cells in vitro are transient, returning to the base line in most cases by 12-24 h) , it is speculated that EMAP II produces multiple effects on inflammatory and vascular cells in the tumor resulting in a sustained perturbation of cellular properties. EMAP II is also likely to have direct effects on neoplastic cells as well, as suggested by the striking decrease of viable cells obtained from murine mammary carcinomas following treatment with EMAP II and TNF.
  • EMAP II induces apoptosis of mammary carcinoma cells as well as other tumor cell lines in vitro. 3 It is unlikely that a tumor would elaborate a mediator designated solely for its destruction, means through which EMAP II could also have a beneficial effect on survival of the neoplasm have been considered.
  • One such mechanism is suggested by the observation that EMAP II induces IL-8 production by MPs. Since IL-8 is a potent inducer of angiogenesis (72, 73), EMAP II may indirectly facilitate the establishment and growth of a tumor early in its development. Further studies with transfected tumor cell lines which overexpress EMAP II, and with reagents which block EMAP II-cellular interactions, will be required to fully assess the many mechanisms likely to be involved.

Abstract

L'invention porte sur un polypeptide purifié d'activation des monocytes endothéliaux (EMAPII) et sur sa méthode d'obtention ainsi que sur des méthodes d'obtention du polypeptide et de détection de ses anticorps. L'invention porte également sur une protéine d'activation des cellules effectrices qui contient une séquence aminoacide homologue du RIGRIVT, et sur sa méthode de détection. L'invention porte enfin sur une méthode de traitement d'une tumeur consistant à administrer au patient une dose efficace dudit polypeptide.
PCT/US1994/011085 1993-09-29 1994-09-29 Polypeptide ii d'activation des monocytes endotheliaux, constituant un mediateur d'activation de la reponse d'hotes WO1995009180A1 (fr)

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AU79615/94A AU7961594A (en) 1993-09-29 1994-09-29 Endothelial monocyte activating polypeptide ii: a mediator which activates host response
JP7510465A JPH09505987A (ja) 1993-09-29 1994-09-29 内皮単球活性化ポリペプチド▲ｉｉ▼：宿主の応答を活性化するメディエーター
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WO1997010841A1 (fr) * 1995-09-18 1997-03-27 The Trustees Of Columbia University In The City Of New York Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux
WO1998008950A1 (fr) * 1996-08-28 1998-03-05 Incyte Pharmaceuticals, Inc. Nouvelle cytokine activant les monocytes
EP0873349A1 (fr) * 1995-06-07 1998-10-28 Human Genome Sciences, Inc. Polypeptide de type iii activateur des monocytes et des cellules endotheliales
US6013483A (en) * 1995-06-07 2000-01-11 Human Genome Sciences, Inc. DNA encoding endothelial monocyte activating polypeptide III
US7264803B2 (en) * 2000-01-19 2007-09-04 Childrens Hospital Los Angeles Methods and pharmaceutical formulations for the treatment of pulmonary hypertension

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EP0873349A1 (fr) * 1995-06-07 1998-10-28 Human Genome Sciences, Inc. Polypeptide de type iii activateur des monocytes et des cellules endotheliales
US6013483A (en) * 1995-06-07 2000-01-11 Human Genome Sciences, Inc. DNA encoding endothelial monocyte activating polypeptide III
EP0873349A4 (fr) * 1995-06-07 2001-03-14 Human Genome Sciences Inc Polypeptide de type iii activateur des monocytes et des cellules endotheliales
US6864226B1 (en) 1995-06-07 2005-03-08 Human Genome Sciences, Inc. Endothelial-monocyte activating polypeptide III
US7045301B2 (en) 1995-06-07 2006-05-16 Human Genome Sciences, Inc. Endothelial-monocyte activating polypeptide III antibodies
US7482326B2 (en) 1995-06-07 2009-01-27 Human Genome Sciences, Inc. Endothelial-monocyte activating polypeptide III
WO1997010841A1 (fr) * 1995-09-18 1997-03-27 The Trustees Of Columbia University In The City Of New York Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux
WO1998008950A1 (fr) * 1996-08-28 1998-03-05 Incyte Pharmaceuticals, Inc. Nouvelle cytokine activant les monocytes
US5885798A (en) * 1996-08-28 1999-03-23 Incyte Pharmaceuticals, Inc. DNA encoding a monocyte activating cytokine
US6090377A (en) * 1996-08-28 2000-07-18 Incyte Pharmaceuticals, Inc. Monocyte activating cytokine
US7264803B2 (en) * 2000-01-19 2007-09-04 Childrens Hospital Los Angeles Methods and pharmaceutical formulations for the treatment of pulmonary hypertension

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CA2172729A1 (fr) 1995-04-06
US6228837B1 (en) 2001-05-08
US6734168B2 (en) 2004-05-11
AU7961594A (en) 1995-04-18
US20020160957A1 (en) 2002-10-31

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